Antitrypanosomal activity of 1,2-dihydroquinolin-6-ols and their ester derivatives

966 J. Med. Chem. 2010, 53, 966–982

DOI: 10.1021/jm900723w

Antitrypanosomal Activity of 1,2-Dihydroquinolin-6-ols and Their Ester Derivatives

Jean Fotie,^{†,^}Marcel Kaiser,^‡Dawn A. Delfın,^†Joshua Manley,^†Carolyn S. Reid,^†Jean-Marc Paris,^§Tanja Wenzler,^‡

´

Louis Maes, Kiran V. Mahasenan,^†Chenglong Li,^†and Karl A. Werbovetz*^,†

^†Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus,

Ohio 43210, ^‡Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, CH-4002 Basel, Switzerland, ^§Laboratoire de

ꢀ

Biochimie, Ecole Nationale Supeꢀrieure de Chimie de Paris, UMR CNRS 7573, 11 Rue Pierre et Marie Curie, 75005 Paris, France, and Faculty of

Pharmaceutical, Biomedical, and Veterinary Sciences, University of Antwerp, Antwerp, Belgium. ^{^}Present address: Department of Chemistry and

Physics, 217 Pursley Hall, Southeastern Louisiana University, Hammond, Louisiana 70402.

Received May 27, 2009

The current chemotherapy for second stage human African trypanosomiasis is unsatisfactory. A

synthetic optimization study based on the lead antitrypanosomal compound 1,2-dihydro-2,2,4-

trimethylquinolin-6-yl 3,5-dimethoxybenzoate (TDR20364, 1a) was undertaken in an attempt to

discover new trypanocides with potent in vivo activity. While 6-ether derivatives were less active than

the lead compound, several N1-substituted derivatives displayed nanomolar IC₅₀values against T. b.

rhodesiense STIB900 in vitro, with selectivity indexes up to >18000. 1-Benzyl-1,2-dihydro-2,2,4-

trimethylquinolin-6-yl acetate (10a) displayed an IC₅₀value of 0.014 μM against these parasites and

a selectivity index of 1700. Intraperitoneal administration of 10a at 50 (mg/kg)/day for 4 days caused a

promising prolongation of lifespan in T. b. brucei STIB795-infected mice (>14 days vs 7.75 days for

untreated controls). Reactive oxygen species were produced when T. b. brucei were exposed to 10a in

vitro, implicating oxidative stress in the trypanocidal mode of action of these 1,2-dihydroquinoline

derivatives.

Introduction

some parts of Africa.⁴Eflornithine is an alternative to mel-

arsoprol treatment for second stage T. b. gambiense infection

but is ineffective against T. b. rhodesiense and must be

administered by slow intravenous infusion in high doses over

a long course.^4,6Recently, eflornithine failure in sub-Saharan

Africa has been observed (Jean Jannin, personal commu-

nication). Eflornithine is currently being studied in combina-

tion with the anti-Chagas disease drug nifurtimox against

second stage infections with T. b. gambiense in the hopes of

extending the usefulness of the former drug.⁷While several

other classes of molecules have displayed activity against

African trypanosomes, no other drugs are approved for use

against second stage disease. A safe, effective, and inexpensive

replacement for melarsoprol and eflornithine against second

stage HAT is desperately needed.

As part of the continuing search for novel entities with

better efficacy and lower toxicity for the treatment of second

stage HAT, a screening program sponsored by the World

Health Organization’s Special Programme for Research and

Training in Tropical Diseases (WHO/TDR) was carried out

at Tibotec Belgium and the Swiss Tropical Institute (STI),

using compounds sourced from Specs (www.specs.net). This

program identified a series of dihydroquinolines with promis-

ing antitrypanosomal activity, of which 1,2-dihydro-2,2,4-

trimethylquinolin-6-yl 3,5-dimethoxybenzoate (TDR20364,

1a) was studied in the greatest detail. This agent displayed

potent and selective antitrypanosomal activity against the T.

b. rhodesiense strain STIB900 (IC₅₀=0.054 μM, SI=300).

However, no cures were achieved and no prolongation of

survival was observed in T. b. brucei (STIB795) infected

NMRI mice treated with 1a at a daily dose of 50 mg/kg/day

Human African trypanosomiasis (HAT^a) or sleeping sick-

ness is caused by Trypanosoma brucei subspecies, parasites

that are transmitted by the tsetse fly. Early symptoms of HAT

include malaise and irregular fevers as well as enlarged lymph

glands and spleen. Parasites are later found in the central

nervous system (CNS), where they cause progressive neuro-

logical disorders ending in death in the absence of chemother-

apy.¹Approximately 17 500 cases of sleeping sickness were

reported for the year 2004, although the disease incidence is

probably 3-4 times higher²(for a review on the current

epidemiological situation, see Simarro et al.³). Pentamidine

and suramin are used to treat the first phase but do not cross

the blood-brain barrier efficiently and thus are not used to

treat second stage disease. Melarsoprol, an organoarsenical, is

used against second stage disease but causes reactive ence-

phalopathy in 5-10% of cases, half of which are fatal.^4,5

Failure rates with melarsoprol have risen to as high as 30% in

*To whom correspondence should be addressed. Phone: (614) 292-

5499. Fax: (614) 292-2435. E-mail: werbovetz.1@osu.edu.

^aAbbreviations: CM-H₂DCFDA, 5-(and-6)-chloromethyl-2⁰,7⁰-di-

chlorodihydrofluorescein diacetate, acetyl ester; CNS, central nervous

system; COSY, correlation spectroscopy; CYP450, cytochrome P450;

DCM, dichloromethane; ESI-MS, electrospray ionization mass spectro-

metry; Et₃N, triethylamine; FDA, Food and Drug Administration;

HAT, human African trypanosomiasis; HMBC, heteronuclear multiple

bond correlation; HMQC, heteronuclear multiple quantum coherence;

HRESI-MS, high resolution electrospray ionization mass spectrometry;

MLSP, melarsoprol; PPT, podophyllotoxin; ROS, reactive oxygen

species; SI, selectivity index; STI, Swiss Tropical Institute; WHO/

TDR, World Health Organization’s Special Programme for Research

and Training in Tropical Diseases.

r

pubs.acs.org/jmc

Published on Web 01/04/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 967

prepared as described for compounds 9a-o and obtained as

brown needles, were stable as free alcohols, while the other

compounds required protection by either acetylation or ben-

zoylation to avoid a color change and to permit satisfactory

elemental analysis. Accordingly, the N1-benzylated deriva-

tives (9a-o), acetyl chloride, and Et₃N were allowed to stir at

room temperature overnight in THF or DCM under nitrogen

to yield 10a-o. Reaction of 1,2-dihydro-2,2,4-trimethylqui-

nolin-6-ols 9a, 9e, and 9f with 3,5-dimethoxybenzoyl chloride

yielded 12a-c, while reaction of 9a with benzoyl chloride, 2-

phenylacetyl chloride, trimethylacetyl chloride, octanoyl chlo-

ride, and cyclohexane carboxylic acid chloride yielded 13a-e,

respectively. The 6-O,N1-diacetylated derivative (15a) and

the N1-methylated 6-O-acetyl analogue (15b) were also pre-

pared. Compound 15a was obtained by heating a mixtureof4,

acetyl chloride, and NaH in THF to reflux to achieve diace-

tylation, while 15b was obtained by heating 1,2-dihydro-2,2,4-

trimethylquinolin-6-yl acetate (14) with iodomethane in re-

fluxing toluene in the presence of Et₃N as base (the structures

for 11a, 11b, 12a-c, 13a-e, 15a, and 15b are in Figure 2).

6-O-BenzylatedandN1-benzylatedderivativescanbeeasily

Figure 1. Structures of 1a, 1b, 2a, and 2b.

ip for 4 days. Nonetheless, the outstanding in vitro antitrypa-

nosomal activity of 1a suggested that this compound could

serve as a hit for the identification of new antitrypanosomal

drug candidates. The compound series was assigned to The

Ohio State University by WHO/TDR for medicinal chemistry

optimization with parasitology support from STI.

We report here initial lead identification studies based on 1a

that resulted in the preparation, characterization, and evalua-

tion of a series of 1,2-dihydroquinoline derivatives for their

antitrypanosomal activity. The in vivo evaluation of some of

thesecompoundsaswell as a preliminaryinvestigation of their

trypanocidal mechanism of action is also described.

differentiated by their H and ¹³C NMR spectra. In the H

NMR spectrum of 6-O-benzylated derivatives, a singlet of one

proton around δ 5.40-5.60 ppm (NH) and another singlet of

two protons around δ 4.90-4.98 ppm (CH₂-O) can be

observed in addition to the aromatic protons, but no signal

around δ 8.30-8.50 ppm attributable to a phenolic hydroxyl

1

Chemistry

Quinolin-6-yl benzoate derivatives 2a and 2b were obtained

by esterification of commercially available 6-hydroxyquino-

line with 3,5-dimethoxybenzoyl chloride (readily prepared by

heating 3,5-dimethoxybenzoic acid to reflux in thionyl

chloride⁸) and with commercially available piperonyloyl

chloride, respectively (see Figure 1 for structures of 1a, 1b,

2a, and 2b). 1,2-Dihydro-2,2,4-trimethylquinolin-6-ol (4) was

obtained by heating commercially available ethoxyquin (3)

to reflux in 48% HBr.⁹Compound 4 was esterified with 3,5-

dimethoxybenzoyl chloride, piperonyloyl chloride, or iso-

phthaloyl dichloride (see Scheme 1) in dichloromethane

(DCM), using triethylamine (Et₃N) as base, to afford com-

pounds 1a, 1b, or 5, respectively.

Compound 4 also served as a versatile precursor for the

synthesis of 1,2-dihydro-2,2,4-trimethylquinoline derivatives

possessing an ether linkage at the 6-position (6a-g, 7) and

N1-benzylated 1,2-dihydro-2,2,4-trimethylquinolin-6-ol deri-

vatives (9a-o). Selective modification of 4 occurs either at the

6-O atom by using an appropriate electrophile in the pre-

sence of NaH/DMF to yield 6-O-benzylated derivatives

(Scheme 2)¹⁰or at N1 with an appropriate electrophile in

the presence of Et₃N/toluene to yield the 6-hydroxy-N1-

substituted derivatives (Scheme 3). This latter reaction was

incomplete when acetone or THF were used as solvent.

Employing more strongly basic conditions (NaH/DMF) af-

fords not only the 6-O-substituted derivatives (70-85%) as

major products but also the corresponding N1,O-disubstitut-

ed compounds (8a-g) as side products (see Scheme 2). Three

of these side products (8e-g) were isolated, characterized, and

tested for their biological activity. Furthermore, the N1-

benzylated compounds were very susceptible to auto-oxida-

tion, as they turned to a dark, sticky gum within a few hours

after preparation regardless of the synthetic and storage

conditions. Despite the fact that a rapid color change was

observed with 9a and that a satisfactory elemental analysis

could not be obtained for this compound, no changes in the

¹H NMR spectrum of 9a were observed when the pure

material was stored at room temperature in an open air

environment for 6 days. Only 11a and 11b, which were

1

group is present. However, the H NMR spectrum of N1-

benzylated derivatives displays a singlet around δ 4.40-

4.60 ppm (CH₂-N) and a hydroxyl resonance (around δ

8.30-8.50 ppm) but no NH signal. These observations were

confirmed by 2D NMR spectroscopy comparing compound

6a as a representative 6-O-benzylated derivative and 11a as a

representative N1-modified derivative. The heteronuclear

multiplebond correlation(HMBC) spectrumof11a displayed

key correlations between the protons at δ 4.41 ppm (benzylic

protons) and the quaternary carbon (C-2) at δ 56.8 ppm and

between the same protons and the aromatic quaternary

carbon (C-8a) at δ 137.2 ppm (see Figure 3). No correlation

was observed between these benzylic protons and the phenolic

carbon (C-6) at δ 148.8 ppm. These data demonstrate that the

methylpyridyl side chain is bound to the N1-atom. Such

correlations between the benzylic protons and C-2 and C-8a

were absent in the HMBC spectrum of 6a. Instead, a correla-

tion between the benzylic protons and the phenolic carbon

at δ 148.5 ppm was observed (see Figure 3), confirming that

the benzyl side chain is linked at the 6-O atom.

Essential precursors 1,2-dihydro-2,2,4-trimethylquinoline

(16a) and 1,2-dihydro-2,2,4-trimethylquinolin-7-ol (16b) were

not commercially available, so these compounds were pre-

paredin our laboratory. Dihydroquinolines have beensynthe-

sized via a variety of methods.^11-14We chose to prepare 16a

and 16b through a modified Skraup cyclization, which in-

volves reaction of the appropriate aniline derivative and a

ketone in the presence of catalytic iodine.^15-18As an adapta-

tion of this method, a mixture of the appropriate aniline

derivative and acetone in toluene was heated to reflux for 24

h in the presenceofacatalytic quantityofiodine toprovide the

corresponding dihydroquinoline. The methyl group on the 7-

O atom (if present) was removed using BBr₃in DCM.^19,20

This was achieved by allowing the reaction mixture initially

maintained at -78 ꢀC to warm to room temperature. N-

Substitution and acetylation were achieved as reported above

to provide 17a and 17b (Scheme 4).

968 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3

Fotie et al.

Scheme 1^a

^aReagents and conditions: (i) HBr, reflux, 16 h; (ii) isophthaloyl dichloride, DCM, Et₃N.

Scheme 2^a

^aReagents and conditions: (i) benzyl bromide derivative, NaH, DMF, room temp, 48 h; (ii) 2-bromoethylbenzene, NaH, DMF, room temp, 48 h.

Scheme 3^a

^aReagents and conditions: (i) benzyl bromide derivative, Et₃N, toluene, reflux, overnight; (ii) CH₃COCl, Et₃N, DCM, room temp, overnight.

The 1,2-dihydro-2,2-dimethylquinoline core was prepared

by reaction between the appropriate aniline derivative and

3-chloro-3-methyl-1-butyne in Et₃N in the presence of copper

metal and copper chloride to yield the corresponding N-(2-

methylbut-3-yn-2-yl)arylamine (18).¹⁸Ring closure was per-

formed in toluene in the presence of copper chloride to

yield the desired 1,2-dihydro-2,2-dimethylquinoline.^18,21,22

O-Dealkylation, N-substitution, and O-acetylation (if

applicable) were achieved using the same conditions as re-

ported above (Scheme 5).

from 7 nM to 89 μM. The two 6-hydroxyquinoline ester

derivatives (2a and 2b) were the least active of the series, with

IC₅₀values of 89 and 80 μM, respectively (Table 1). Among

the 1,2-dihydroquinolines, the 6-ether (6a-g, 7) and the N1-

benzylated 6-ether (8e-g) derivatives were far less active than

the 6-esters or their N1-benzylated counterparts. The 6-ether

derivatives displayed IC₅₀values ranging from 3.7 μM (for 7)

to 13 μM (for 6b), while the N1-benzylated 6-ether derivatives

(8e-g) were much less active (IC₅₀values of 86 and 63 μM for

8g and 8e, respectively) except for 8f which displayed an IC₅₀

value of 3.6 μM (Table 2). However, all the 1,2-dihydroquino-

line ester derivatives (1a-b, 5; see Table 1), the N1-benzylated

6-hydroxy compounds (9a-b, e-f, n, 11a,b, 20a), and their

ester derivatives (10a-o, 12a-c, 13a-e and 20b) were among

Antitrypanosomal Activity

A total of 53 compounds (1a-20b) were evaluated in vitro

for their antitrypanosomal activity and cytotoxicity. The

results of the testing against T. b. rhodesiense bloodstream form

trypomastigotes (STIB900) as well as the assessment of the

cytotoxicity of compounds 1a-20b against rat myoblast cells

(L6) are summarized in Tables 1-4, with melarsoprol and

podophyllotoxin used as the reference agents. Compounds

1a-20b displayed differentlevels ofantitrypanosomalactivity

against T. b. rhodesiense (STIB900), with IC₅₀values ranging

the most active agents (Tables 3 and 4). Compound 5 (IC₅₀

<

0.008 μM) was the most potent of the ester derivatives,

while 9a (IC₅₀= 0.007 μM) was the most potent of the N1-

benzylated congeners. The 6-O-acetyl and 6-O-benzoyl deri-

vatives of 9a, compounds 10a (IC₅₀= 0.014 μM) and 13a

(IC₅₀= 0.013 μM), were the most potent of the 6-O-acyl N1-

benzylated derivatives. Interestingly, any substitution on the

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 969

Scheme 4^a

^aReagents and conditions: (i) I₂, toluene, reflux, 24 h; (ii) BBr₃, DCM

(-78 to 25 ꢀC), if applicable; (iii) benzyl bromide, Et₃N, reflux, over-

night; (iv) CH₃COCl, Et₃N, DCM, room temp, overnight, if applicable.

Scheme 5^a

Figure 2. Structures of 11a, 11b, 12a-c, 13a-e, 15a, and 15b.

Figure 3. Key HMBC correlations for 11a and 6a.

^aReagents and conditions: (i) 3-chloro-3-methyl-1-butyne, Cu, CuCl

(10%/10% w/v), Et₃N, room temp; (ii) CuCl (10%), toluene, reflux; (iii)

HBr, reflux, 16 h; (iv) ArCH₂Br, Et₃N, reflux, overnight; (v) CH₃COCl,

Et₃N, DCM, if applicable.

phenyl ringof the N1-benzyl group resulted ina lossofactivity

(compare 9a with 9b, 9e, 9f, 9n and compare 10a with 10b-o),

albeit this was a minor loss in some cases. The identity of the

ester group atthe6-O atom hadlittleeffect on activity, asthere

was only a 2-fold difference in potency among N1-benzylated

esters 10a, 12a, and 13a-e. The 6-O-acetyl N1-acetyl analogue

(15a, IC₅₀=5.3μM) andthe 6-O-acetyl-N1-methylderivative

(15b, IC₅₀= 1.4 μM) exhibited reduced activity compared to

all of the N1-benzylated derivatives possessing an OH group

at position 6 or their esterified counterparts. The nor-hydroxy

derivative 1-benzyl-1,2-dihydro-2,2,4-trimethylquinoline (17a,

IC₅₀=57 μM) displayed weak activity. 1-Benzyl-1,2-dihydro-

2,2,4-trimethylquinolin-7-yl acetate (17b, IC₅₀4.4 μM), the

7-O-acetyl derivative, was less potent than the 6-O-acetyl com-

pounds (although it was more active than nor-hydroxy con-

gener 17a).

many of these compounds displayed SI values exceeding

1000 (5, SI > 7800; 10a, SI = 1700; 12b, SI > 3900; 12c, SI >

6000; 13a, SI>18 000; 13b, SI=10 000; 13d, SI=13000; 13e,

SI > 8200).

In Vivo Antitrypanosomal Activity

Based on the potent in vitro antitrypanosomal activity and

selectivity of the N1-benzyl dihydroquinoline 6-esters against

T. b. rhodesiense, compounds 10a, 10d, and 10m were selected

for evaluation in a murine model of African trypanosomiasis.

These agents were administered to T. b. brucei (STIB795)

infected mice by intraperitoneal injection at a daily dose of 50

mg/kg for 4 days. The results of this in vivo evaluation,

reported in Table 5, revealed that 10a was the most active

compound. While all animals in the other groups had died

because of infection by day 14, each of the mice receiving

compound 10a was still alive at this point. Nonetheless, mice in

this group relapsed by day 10 (were positive for parasites) and

were euthanized on day 14 because of animal welfare regulations.

Compounds 10d and 10m were substantially less active than 10a,

and the survival times of these groups were not statistically

different from that of the control group. No overt signs of toxicity

related to the compounds were noted in any of the mice.

Cytotoxicity and Selectivity

Tables 1-4 also summarize the in vitro toxicities of the

compounds to L6 rat myoblasts and their in vitro antitrypa-

nosomal selectivity indexes (SI) relative to L6 cells, expressed

as the ratio IC₅₀(L6)/IC₅₀(T. b. rhodesiense). The 6-O-benzy-

lated dihydroquinolines (compounds 6a-g, IC₅₀= 45-77

μM) and their N1,O-disubstituted congeners (compounds

8e-g, IC₅₀g 180 μM) were less toxic to L6 myoblasts than

either the N1-benzylated 6-hydroxydihydroquinolines (com-

pounds 9a,b, 9e,f, and 9n, IC₅₀= 6.2-11 μM) or the 6-O-

acetyl-N1-benzyl derivatives (compounds 10a-o, IC₅₀= 9-

31 μM). Acetylation of 9a,b, 9e,f, and 9n to give 10a,b, 10e,f,

and 10n resulted in at least a 2-fold decrease in cytotoxicity.

With the exception of 12a (IC₅₀= 24 μM), replacement of

the methyl ester present in compounds 10a-n with a larger

ester substituent resulted in a decrease in cytotoxicity

(compounds 12b,c and 13a-e, IC₅₀g 160 μM). Most notably,

Mode of Action

Since we suspected reactive oxygen species (ROS) to be

involved in the mechanism of action of these agents, 10a, its 7-

O-acetyl counterpart 17b, and the nor-hydroxy compound

970 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3

Fotie et al.

Table 1. In Vitro Antrypanosomal Activity and Cytotoxicity of Compounds 1a-5

^aMLSP, melarsoprol. ^bPPT, podophyllotoxin. ^cTrypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{23 d}Cytotoxicity

(L6 rat myoblast cells). Average of duplicate determinations.^{24 e}Selectivity index expressed as the ratio IC₅₀(L6)/IC₅₀(T. b. rhodesiense). ^fCalculated

using ChemDraw Pro 11.0.1 (CambridgeSoft).

17a were evaluated for their abilityto generate ROS withinthe

parasite. Our assay to detect ROS in T. b. brucei in vitro

employed

H₂O₂showed only a 3.3-fold increase in fluorescence com-

pared to the negative control. Compound 17a, which lacks a

hydroxyl group, and 17b, which possesses a 7-acetoxy group,

cause no increase in fluorescence when trypanosomes were

exposed to 0.1, 1, and 10 μM concentrations of these agents.

5-(and-6)-chloromethyl-2⁰,7⁰-dichlorodihydro-

fluorescein diacetate, acetyl ester (CM-H₂DCFDA), which

has been used previously to assess cellular ROS levels.^25,26

CM-H₂DCFDA diffuses through membranes during cell

loading. Then intracellular esterases produce the correspond-

ing carboxy derivative, which oxidizes to a highly fluorescent

derivative in the presence of reactive oxygen species. Figure 4

shows the results obtained from the evaluation of 10a, 17a,

and 17b compared to H₂O₂and 1% DMSO used as positive

and negative controls, respectively. Compound 10a, which

bears a 6-acetoxy group, caused a concentration-dependent

increase in fluorescence in trypanosome-containing samples

incubated with this compound for 24 h. A slight increase in

fluorescence was observed when parasites were exposed to

0.1 μM 10a, while fluorescence levels were almost 4-fold higher

than negative control samples when trypanosomes were

incubated with 10 μM 10a. T. b. brucei exposed to 250 μM

Discussion

As part of a continuing search for novel entities with better

efficacy and lower toxicity for the treatment of late stage

HAT, a screening program supported by WHO/TDR identi-

fied a promising series of 1,2-dihydroquinolines with antitry-

panosomal activity. While in vitro testing of 1a against T. b.

rhodesiense (STIB900) revealed the potent activity of this

compound (IC₅₀= 0.054 μM), it was inactive in a murine

T. b. brucei model. Subsequent studies described here have

identified dihydroquinolines with potent and selective in vitro

antitrypanosomal activity, and 10a displays promising in vivo

activity in an animal model of African trypanosomiasis.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 971

Table 2. In Vitro Antrypanosomal Activity and Cytotoxicity of Ethers 6a-8g

IC₅₀, μM

compd

R₁

R₂

T. b. rhodesiense^c

cytotoxicity^d

SI ^e

14

ClogP ^f

6a

6b

H

benzyl

4.1

13

56

68

4.06

3.81

3.94

4.98

4.34

6.08

8.18

-

3,5-dimethoxybenzyl

4-methoxybenzyl

2-methoxybenzyl

5

6c

6d

5.2

5.3

11

77

48

15

9

6e

3-methoxybenzyl

61

45

5

6f

6g

3-trifluoromethylbenzyl

4-trifluoromethylbenzyl

phenethyl

8.8

4.5

3.7

63

5

48

83

11

22

>3

>50

>2

1000

-

7

8e

3-methoxybenzyl

3-trifluoromethylbenzyl

4-trifluoromethylbenzyl

3-methoxybenzyl

3-trifluoromethylbenzyl

4-trifluoromethylbenzyl

>210

>180

7.9

8f

8g

MLSP^a

PPT^b

3.6

86

0.008

-

0.012

-

^aMLSP, melarsoprol. ^bPPT, podophyllotoxin. ^cTrypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{23 d}Cytotoxicity

(L6 rat myoblast cells). Average of duplicate determinations.^{24 e}Selectivity index expressed as the ratio IC₅₀(L6)/IC₅₀(T. b. rhodesiense). ^fCalculated

using ChemDraw Pro 11.0.1 (CambridgeSoft).

Dihydroquinolines are mainly known for their antioxidant

activity,^27-31although they have also been reported to possess

anti-inflammatory,^32,33fungicidal,³⁴antiatherosclerotic,³⁵

and hormone receptor modulator^36,37properties. However,

compound 3, also known as ethoxyquin, remains the most

well-knownmember ofthis classofmolecules. Compound3, a

Food and Drug Administration (FDA) approved antioxidant

commonly used as a preservative in the food processing

industry,^38,39is a fat stabilizer and preservative found primar-

ily in premium pet foods containing high fat levels. It has been

used for over 30 years to prevent oxidation and rancidity in

fats in order to maintain the wholesomeness and the quality in

pet foods. The FDA has also approved its use as food additive

in the production of paprika, chili powder, and ground chili.

Studies have shown that 3 displays a cancer chemoprotective

effect by inducing detoxification enzymes such as glutathione

S-transferase⁴⁰and aldehyde reductase.^41,42Since 3 is an

FDA-approved food additive and the related compounds

studied here possess high selectivity indexes, the further

investigation of these agents as antitrypanosomal drug candi-

dates is warranted.

The fact that esterified 6-hydroxyquinoline compounds 2a

and 2b were inactive (IC₅₀of 89 and 80 μM, respectively)

against T. b. rhodesiense in vitro while the 1,2-dihydroquino-

line derivatives (1a and 1b) bearing the same ester side chains,

respectively, displayed good activities (IC₅₀of 0.054 and

0.30 μM, respectively) suggests that the dihydroquinoline core

is required for antitrypanosomal activity. This hypothesis was

further supported by the observation that 4, the hydroxy

derivative of the initial lead compound 1a, displayed signifi-

cant antitrypanosomal activity (IC₅₀= 0.78 μM), while its

isophthalate dimer derivative (5) exhibited outstanding po-

tency (IC₅₀< 0.008 μM). It is also important to note that 4

was less active than any of the ester derivatives, possibly

because of its higher hydrophilicity that may restrict passive

diffusion of the compound into the parasite. The calculated

log P (ClogP) values for 4, 15a, and 15b are lower than

the corresponding values for other dihydroquinoline-6-

esters examined in this study (see Tables 1, 3, and 4), provi-

ding further support for this hypothesis. This could also

explain why the pyridylmethyl derivatives 11a and 11b

were generally less active than the N1-benzyl derivatives

10a-o, 12a-c, and 13a-e. A scatter plot of IC₅₀values vs

ClogP for all monomeric dihydroquinolines containing a

6-hydroxyl group or a 6-O-acylated function (compounds

1a,b, 9a,b, 9e,f, 9n, 10a-o, 11a,b, 12a-c, 13a-e, 15a,b,

and 20a,b; see Supporting Information Figure S1) indicates

that such agents with ClogP values less than 4 tend to possess

weaker in vitro antitrypanosomal potency than those with

ClogP values of 4 or greater. The hydrophilicity of 4 might

also explain why the parent compound (1a) was inactive in the

murine trypanosomiasis model. In vivo, rapid conversion

of 1a to 4 is likely to occur through the action of host esterases.

This latter compoundmay beless likely toenter the parasiteor

could have been excreted rapidly from the animal (perhaps

aided by phase 2 metabolism). Thus, although compound 5

exhibitedpotent in vitro antitrypanosomal activity, wedid not

pursue the in vivo evaluation of this compound because of our

belief that 4 would be rapidly generated in the animal model.

A series of 1,2-dihydroquinolines possessing side chains

that would be less susceptible to rapid host metabolism was

also prepared. This was achieved by attaching benzyl side

chains at the 6-O-position, at the N1-atom, or at both posi-

tions to generate a series of 6-O-benzylated ether (6a-g), N1-

benzylated (9a-o, 11a,b), or 6-O,N1-dibenzylated (8e-g)

derivatives. A benzyl side chain was chosen both to mimic

the side chain present in the parent compound and to intro-

duce a groupwhere several analogues could be prepared to aid

in further developing a structure-activity relationship. In the

in vitro assay against T. b. rhodesiense, all the 6-ether com-

pounds (6a-g, 7) displayed reduced activity (IC₅₀= 3.7-13

μM) compared to the lead compound 1a (IC₅₀= 0.054 μM),

while all the 6-O,N1-dibenzylated derivatives (8e-g) were

very weak except for 8f (3.6 μM), a compound that exhibited

972 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3

Fotie et al.

Table 3. In Vitro Antrypanosomal Activity and Cytotoxicity of Compounds 9a-13e

IC₅₀, μM

T. b. rhodesiense^c

compd

R₁

R₂

cytotoxicity^d

SI ^e

970

ClogP ^f

9a

9b

benzyl

H

0.007

0.14

6.8

11

4.34

4.09

4.90

4.83

4.21

4.32

4.06

4.35

4.87

4.8

3,5-dimethoxybenzyl

4-chlorobenzyl

4-methylbenzyl

4-methoxybenzyl

benzyl

H

83

130

9e

H

0.061

0.058

0.17

7.9

8.1

6.2

24

9f

9n

H

140

37

H

10a

10b

10c

10d

10e

10f

10g

10h

10i

acetyl

H

0.014

0.10

0.26

1700

240

3,5-dimethoxybenzyl

4-cyanobenzyl

3-cyanobenzyl

4-chlorobenzyl

4-methylbenzyl

3,4-dichlorobenzyl

3-chlorobenzyl

3-trifluoromethyl-benzyl

4-trifluoromethylbenzyl

3-methylbenzyl

3-methoxybenzyl

4-fluorobenzyl

4-methoxybenzyl

4-tert-butylbenzyl

2-pyridylmethyl

4-pyridylmethyl

benzyl

25

29

110

170

0.18

31

22

120

54

0.39

21

28

0.053

0.039

0.041

0.36

530

740

5.43

4.87

5.24

4.8

29

27

660

39

10j

14

28

10k

10l

0.34

0.32

82

91

29

4.19

4.47

4.19

6.02

3.40

2.98

5.96

6.52

6.45

6.21

6.16

6.24

7.06

6.29

-

10m

10n

10o

11a

11b

12a

12b

12c

13a

13b

13c

13d

13e

MLSP^a

PPT^b

0.041

0.048

0.67

710

310

15

9

18

13

17

1.10

0.34

H

13

24

39

3,5-dimethoxybenzoyl

benzoyl

0.023

0.048

0.033

0.013

0.015

0.028

0.017

0.028

0.008

-

1000

>3900

>6000

>18000

11000

6000

13000

>8200

1000

-

4-chlorobenzyl

4-methylbenzyl

benzyl

>190

>200

>240

160

170

220

>230

7.9

0.012

benzyl

phenylacetyl

trimethylacetyl

octanoyl

cyclohexanecarbonyl

benzyl

-

^aMLSP, melarsoprol. ^bPPT, podophyllotoxin. ^cTrypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{23 d}Cytotoxicity

(L6 rat myoblast cells). Average of duplicate determinations.^{24 e}Selectivity index expressed as the ratio IC₅₀(L6)/IC₅₀(T. b. rhodesiense). ^fCalculated

using ChemDraw Pro 11.0.1 (CambridgeSoft).

unexplained but nonetheless modest potency. On the other

hand, the 6-hydroxy-N1-benzylated compounds (9a,b, 9e,f,

9n) possessed in vitro antitrypanosomal activities (IC₅₀=

Substitution of the phenyl ring present in the N1-benzyl group

resulted in a decrease in antitrypanosomal activity, as com-

pounds 10b-o were 3.3- to 56-fold less active than 10a. There

is no clear antitrypanosomal structure-activity relationship

for these phenyl substitutions, as some meta- and para-sub-

stituted compounds show potent activity (10g-i, 10m, 10n,

with IC₅₀values from 0.039 to 0.051 μM) while others do not

(such as 10e and 10l, with IC₅₀values of 0.18 and 0.32 μM,

respectively). Nonetheless, all of these compounds possessed

submicromolar IC₅₀valuesandwere substantiallymore active

than the 6-O-benzylated ethers and 6-O,N1-dibenzylated

(8e-g) derivatives.

Furthermore, 15a and 15b, two 6-acetoxy derivatives carry-

ing acetyl and methyl groups at the N1 position, respectively,

displayed only marginal activity (IC₅₀of 5.3 and 1.4 μM,

respectively), suggesting an important role for the N1-benzyl

side chain on the antitrypanosomal activity of these com-

pounds. Removing the methyl group at the 4-position of the

dihydroquinoline ring (20a and 20b, IC₅₀of 0.13 and 0.20 μM,

respectively) decreased the activity of these compounds com-

paredtotheir4-methyl counterparts(9a and 10b, IC₅₀of0.007

0.007-0.17 μM) that were equally potent or superior to that

of the parent compound 1a. Compound 9a was the most

potent of the series, with an IC₅₀of 0.007 μM. However, as

mentioned previously, these N1-benzylated compounds were

susceptible to auto-oxidation except for 11a,b and 20a. This

type of behavior has been reported previously for 5-hydroxy-

diclofenac, a p-hydroxyaniline containing metabolite of the

anti-inflammatory drug diclofenac. 5-Hydroxydiclofenac is

believed to undergo auto-oxidation because of its ability to

form glutathione conjugates in the absence of enzymatic

activation.^43,44Thus, the unstable alcohols obtained in our

studies were protected through esterification to generate

prodrugs (10a-o, 12a-c, 13a-e). These compounds were

prepared because we hypothesized that the ester function

would be hydrolyzed both in vitro and in vivo to generate

the active species. This strategy was successful in that all

the 6-O-acylated, N1-benzylated congeners displayed excel-

lent antitrypanosomal activity (IC₅₀= 0.013-0.67 μM).

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 973

Table 4. In Vitro Antrypanosomal Activity and Cytotoxicity of Compounds 15a-20b

IC₅₀, μM

compd

R₁

R₂

R₃

R₄

T. b. rhodesiense^c

cytotoxicity^d

SI ^e

29

ClogP ^f

15a

15b

acetyl

Me

H

acetoxy

H

Me

H

5.3

1.4

160

41

1.69

2.83

4.73

4.32

4.16

3.89

-

29

2

17a

17b

benzyl

57

110

71

acetoxy

H

OH

4.4

16

42

20a

20b

MLSP^a

PPT^b

H

0.13

0.20

0.008

-

5.5

10

3,5-dimethoxy-benzyl

acetoxy

H

50

7.9

0.012

1000

-

^aMLSP, melarsoprol. ^bPPT, podophyllotoxin. ^cTrypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{23 d}Cytotoxicity

(L6 rat myoblast cells). Average of duplicate determinations.^{24 e}Selectivity index expressed as the ratio IC₅₀(L6)/IC₅₀(T. b. rhodesiense). ^fCalculated

using ChemDraw Pro 11.0.1 (CambridgeSoft).

Table 5. Activity of 10a, 10d, and 10m against T. b. brucei STIB795 in Mice^a

compd

dose ((mg/kg)/day), route

formulation

mean survival time ( standard deviation (day)

control

10a

10d

10% DMSO

7.75 ( 3.50

>14^b

4 ꢀ 50, ip

9.25 ( 2.22

8.75 ( 3.78

10m

^aThe veterinary reference drug diminazene typically cures animals in this assay (no parasitemia detected 30 days after infection) when given at 4 ꢀ 10

(mg/kg)/day ip. ^bMice relapsed by day 10 and were euthanized on day 14 after infection; p > 0.05 compared to the control group.

and 0.014 μM, respectively). The N1-benzylated derivatives

carrying the same ester side chain as the parent com-

pound (12a-c) also displayed very good in vitro potency

(IC₅₀of 0.023, 0.048, and 0.033 μM, respectively). Com-

pounds carrying aliphatic (13c-e, IC₅₀of 0.028, 0.017, and

0.028 μM, respectively) or a 2-phenylacetyl (13b, IC₅₀of

0.015 μM) ester side chains were as potent and selective as

their benzoylated or acetylated counterparts, with no major

differences in activity among molecules possessing the various

side chains.

Scheme 6^a

Perhaps the most striking observation from this structure-

activity relationship investigation is the fact that com-

pounds lacking the 6-oxygen atom altogether (17a) or

bearing an oxygen atom at the 7-position rather than the 6-

position (17b) were far less potent than those containing an

alcohol or acyloxy group at the 6-position. These key ob-

servations highlight the importance of a 6-oxygen atom for

antitrypanosomal activity in this series of compounds and

serve as the foundationfor our mechanistic hypothesis. Onthe

basis of the in vitro antitrypanosomal structure-activity

relationship and previous literature concerning ethoxyquin

cited below, we hypothesize that the antitrypanosomal 1,2-

dihydroquinolines increase the oxidative stress on the para-

site, with a free hydroxyl group at the 6-position being critical

for activity. We propose that compounds esterified at the

6-oxygen atom are hydrolyzed rapidly by trypanosomal

esterases, followed by two-electron oxidation to a quinone

imine species.^28,45This quinone imine could then undergo a

single electron reduction to form a semiquinone that may

enhance the rate of formation of reactive oxygen species such

as superoxide and/or other free radicals.^46,47The added strain

on the parasite’s antioxidant machinery could lead to lethal

consequences for the organism (see Scheme 6). However, 17a

^aProposed antitrypanosomal mechanism of 2,2,4-trimethyl-1,2-di-

hydroquinolines.

and 17b, molecules that cannot form a quinone imine inter-

mediate and cause little or no production of reactive oxygen

species in the parasite, are far less potent against trypano-

somes in vitro. Quinones are known to be substrates for

trypanothione reductase,^46,48and this enzyme is a potential

target of these 1,2-dihydroquinoline derivatives. This hypoth-

esis merits further investigation.

When comparing the antioxidant effect of ethoxyquin with

that of some of its derivatives, De Koning and Milkovitch

found that 1,2-dihydroquinolines that lack a hydroxyl or

alkoxyl group at the 6-position of their core (even with such

a group at the 8 position) displayed little or no antioxidant

properties.⁴⁹Thorisson et al. found that a quinone imine

derivative of ethoxyquin displayed significant antioxidant

974 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3

Fotie et al.

Figure 4. Evaluation of the capacity of compounds 10a, 17a, and 17b to generate reactive oxygen species in T. b. brucei using a CM-H₂DCFDA

fluorescence assay. Parasites loaded with CM-H₂DCFDA were incubated at 37 ꢀC for 24 h with or without test compounds at the

concentrations indicated, and fluorescence intensity was measured by flow cytometry as indicated in the Experimental Section. The results

shown represent the mean ( standard error obtained in three independent experiments.

activity.²⁸These authors hypothesized that this activity could

be due to the ability of the quinone imine species to act as a

radical acceptor. Hayes et al. later postulated that the anti-

cancer chemopreventive effect of ethoxyquin stems from the

fact that ethoxyquin is metabolized in vivo to dihydroquino-

lin-6-ol 4 (see Figure 1) by CYP450 isoenzymes, and this latter

compound displayed a chemopreventive effect through its

oxidation toa quinoneimine.⁵⁰Atfirst glance, the antioxidant

properties of the quinone imine observed in earlier chemo-

prevention studies seem to contradict our hypothesis that

these trypanocidal molecules act by increasing oxidative stress

in the parasite. However, given that trypanothione reductase

carries out the single electron reduction of quinones and

naphthoquinones,^46,48the quinone imine may be handled

similarly by the parasite enzyme and could be the source of

significant amounts of reactive oxygen species in the trypano-

some. To examine whether the quinone imine form of 9a was

stabilized compared to other compounds of lesser trypanoci-

dal potency, quantum chemical calculations were performed

for 9a along with dihydroquinolin-6-ol 4, ether 6a, and

the dihydroquinolin-6-ols corresponding to 15a and 15b (see

Supporting Information Table S1). Compounds 6a and the

dihydroquinolin-6-ol corresponding to 15a clearly show

the least potential to be oxidized to the quinone imine since

(1) their HOMO orbitals gain the least stability energy during

oxidation and (2) their overall solution phase energies show

the greatest increase during oxidation. These electronic data

helprationalizethe poor antitrypanosomal activities of6aand

15a. However, these calculations do not allow us to distin-

guish between 4, 9a, and the dihydroquinolin-6-ol corres-

ponding to 15b. Since the ClogP values of 4 and 15b are

significantly lower than that of 10a and its corresponding

dihydroquinolin-6-ol 9a as mentioned earlier, lipophilicity is

likely to play an important role in trypanocidal potency.

While concentrations of 10a above the IC₅₀value of this

compound are required to detect elevated levels of reactive

oxygen species in trypanosomes (Figure 4), redox cycling may

still be responsible for the trypanotoxic effect of 10a. Since

quinones can act as subversive substrates for trypanosomatid

trypanothione reductase in vitro and result in oxygen con-

sumption,⁴⁶redox cycling in live trypanosomes with the

quinone imine formed from 10a would generate superoxide

that could be reduced by trypanosome superoxide dismu-

tase,⁵¹thus preventing the detection of ROS at low concen-

trations of 10a. Such levels of 10a may still be toxic, however,

because of the consumption of reducing equivalents that

would occur during futile redox cycling of the quinone imine.

At higher concentrations of 10a, the trypanosome superoxide

dismutase may be overwhelmed, resulting in the detection of

ROS. Another possible explanation for the relatively low

levels of ROS observed at 100 nM 10a could be that the

ROS determination was made after 24 h of exposure to 10a,

while the IC₅₀value shown in Table 3 was measured after a

70 h incubation of trypanosomes with 10a. The observed

structure-activity relationship is also consistent with attack

on the quinone imine intermediate by a cellular nucleophile,

resulting in the death of the parasite through interference with

a critical process within the trypanosome or the formation of

lethal adducts. However, given the importance of trypa-

nothione reductase to trypanosomatids, the ability of related

quinones and naphthoquinones to act as subversive substrates

for this enzyme,^46,48and the high selectivity of these dihydro-

quinolines for T. brucei compared to L6 cells (Table 1), we feel

that the quinone imine intermediate shown in Scheme 6

is unlikely to exert trypanocidal effects by acting as an electro-

phile.

Quinone imine metabolites of drugs such as acetaminophen

and diclofenac are nonetheless believed to cause hepatotoxi-

city due to their reactivity with cellular nucleophiles.^52-54A

potential strategy to decrease the risk of hepatic injury due to

administration of these dihydroquinoline compounds for the

treatment of HAT is to develop ester-containing derivatives

that are hydrolyzed specifically by trypanosomal esterases,

thus restricting the generation of the quinone imine species to

the site of infection. Also, acetaminophen-induced liver injury

is commonly treated by the administration of N-acetylcys-

teine,⁵⁵which reverses depletion of glutathione levels.⁵⁶Con-

sidering the severity of second stage HAT, the lack of

appropriate treatments for this disease, and the potent in vitro

activity of these simple dihydroquinolines, investigation of

this class of antitrypanosomal molecules should continue

while carefully monitoring the potential toxicity of a quinone

imine intermediate.

Conclusion

HAT is still a huge problem in sub-Saharan Africa. The

disease causes much physical and economic hardship, parti-

cularly in rural communities that are affected by the tsetse

vector and that do not have easy access to modern medical

facilities. The current drugs used to treat second stage HAT

are losing their effectiveness, most likely because of the

emergence of drug-resistant parasites. We report here the

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 975

synthesis of a series of 1,2-dihydroquinolines with nanomolar

in vitro antitrypanosomal potency. Prodrug 10a extended the

lifespan of mice infected with T. b. brucei, indicating that

further investigation of this class of molecules as potential

candidates against HAT is essential. Future studies will focus

on preparing potent, metabolically stable analogues posses-

sing properties that will allow the compounds to cross the

blood-brain barrier and thus display efficacy against second

stage HAT. Efforts will also be undertaken to further eluci-

date the metabolism, pharmacokinetics, and the antitrypano-

somal mechanism of action of this novel and promising class

of compounds.

Quinolin-6-yl Benzo[d][1,3]dioxole-5-carboxylate (2b). The

reaction was similar to the one above using 6-hydroxyquinoline

(300 mg, 2.07 mmol) and piperonyloyl chloride (382 mg, 2.07

mmol) to yield 2b (326 mg, 54%). Purification was carried out

on a silica gel column using hexanes-ethyl acetate (3:7) to

provide the desired material as a white powder, mp 153-

155 ꢀC. ¹H NMR (CDCl₃): δ 6.09 (2H, s), 6.93 (1H, d, J=8.3

Hz), 7.42 (1H, m), 7.57 (1H, dd, J₁= 9.3 and J₂=2.1 Hz), 7.65

(1H, s), 7.68 (1H, d, J=2.1 Hz), 7.87 (1H, d, J = 8.3 Hz), 8.15

(2H, m), 8.92 (1H, d, J=3.6 Hz). ¹³C NMR (CDCl₃): δ 102.1,

108.2, 110.0, 118.6, 121.6, 123.1, 124.9, 126.4, 128.6, 131.1,

135.8, 146.4, 148.0, 148.8, 150.2, 152.4, 164.5. ESI-MS: [M þ

H]^þm/z 294, [M þ Na]^þm/z 316. HRESI-MS: [M þ H]^þm/z

294.0770 (calcd 294.0776). Anal. (C₁₇H₁₁NO₄) C, H, N.

1,2-Dihydro-2,2,4-trimethylquinolin-6-ol (4). Ethoxyquin was

suspended in 48% HBr and heated to reflux for 16 h. The

mixture was cooled to room temperature and partitioned be-

tween chloroform and aqueous Na₂CO₃(1 M).⁹The organic

layer was further washed with water, concentrated under re-

duced pressure, and purified on a silica gel column using

hexanes-ethyl acetate (7:3) to yield 4 (47%) as a yellow powder,

Experimental Section

General Experimental. H NMR (300 MHz), ¹³C NMR (75

1

MHz), correlation spectroscopy (COSY), heteronuclear multi-

ple quantum coherence (HMQC), and HMBC spectra were

recorded on a Bruker 300 UltraShield spectrometer. Electro-

spray ionization mass spectrometry (ESI-MS) and high resolu-

tion ESI-MS (HRESI-MS) were performed by The Ohio State

University Mass Spectrometry and Proteomics Facility. Melting

points were measured on a Thomas-Hoover capillary melting

point apparatus and are uncorrected. Anhydrous DCM and

toluene were distilled over P₂O₅or CaCl₂and stored under

1

mp 178-179 ꢀC (lit.⁴⁴180-181 ꢀC). H NMR (DMSO-d₆): δ

1.33 (6H, s), 1.99 (3H, s), 5.78 (1H, s), 6.82 (1H, d, J=8.3 Hz),

6.86 (1H, s), 7.38 (1H, d, J = 8.3 Hz), 9.96 (1H, brs, NH), 10.87

(1H, brs, OH). Anal. (C₁₂H₁₅NO) C, H, N.

1,2-Dihydro-2,2,4-trimethylquinolin-6-yl 3,5-Dimethoxy-

benzoate (1a). The reaction was similar to the preparation of

2a using compound 4 (600 mg, 3.2 mmol) and 3,5-dimethox-

ybenzoyl chloride (642 mg, 3.2 mmol) to yield, after purification

on a silica gel column using hexanes-ethyl acetate (8:2), 1a (669

mg, 59%) as a yellow powder. This compound was further

recrystallized from hexanes-ethyl acetate-dichloromethane

(2:2:1) to yield yellow needles, mp 130-132 ꢀC. ¹H NMR

(CDCl₃): δ 1.30 (6H, s), 1.97 (3H, s), 3.86 (6H, s), 5.37 (1H, s),

6.46 (1H, d, J = 8.4 Hz), 6.71 (1H, s), 6.83 (1H, dd, J₁= 8.4 and

J₂= 2.4 Hz), 6.89 (1H, d, J = 2.4 Hz), 7.34 (1H, s), 7.35 (1H, s).

¹³C NMR (CDCl₃): δ 18.7, 31.1, 52.2, 55.8, 106.4, 107.8, 113.2,

116.8, 121.2, 122.5, 128.3, 129.5, 132.0, 141.4, 142.3, 160.9,

165.8. ESI-MS: [M þ H]^þm/z 354, [M þ Na]^þm/z 376.

HRESI-MS: m/z 354.1711 (calcd 354.1705). Anal. (C₂₁H₂₃-

NO₄) C, H, N.

1

molecular sieves (type 4A, /₁₆in. pellets). THF was distilled

over Na metal and used immediately after collection. Reaction

mixtures were monitored using TLC silica gel 60 F₂₅₄plates

from EMD Chemicals Inc. Gravity and flash column chroma-

tography were performed using type 60A silica gel (60-230

mesh) from Fisher Scientific. All the compounds were further

purified using silica gel GF preparative 1000 μm UV₂₅₄plates

from Analtech. As described in the text, N1-benzyl-2,2,4-tri-

methyldihydroquinolin-6-ols were susceptible to oxidation and

were converted to their stable ester derivatives. All other target

compounds displayed a purity of g95% (elemental analyses

within 0.4% of theoretical values) unless otherwise noted.

Elemental analyses were performed by Atlantic Microlabs,

Norcross, GA.

Materials. All chemicals and solvents were purchased from

Aldrich Chemical Co., Fisher Scientific, or Acros Organics and

were used without further purification unless stated otherwise.

Preparation and Characterization of Compounds. Preparation

of 6-O-Acyl Derivatives. Ester compounds were prepared by

allowing a mixture of either 6-hydroxyquinoline or 1,2-dihydro-

2,2,4-trimethylquinolin-6-ol (4) and the appropriated acyl

chloride in DCM or THF to stir overnight in the presence

of Et₃N. While 6-hydroxyquinoline is commercially avail-

able, 1,2-dihydro-2,2,4-trimethylquinolin-6-ol was obtained by

heating the commercially available ethoxyquin to reflux in 48%

HBr for 16 h.⁹Many of the acyl chlorides used were com-

mercially available. Those that were not were prepared by

heating the corresponding carboxylic acid to reflux in thionyl

chloride.⁸

1,2-Dihydro-2,2,4-trimethylquinolin-6-yl Benzo[d][1,3]dioxole-

5-carboxylate (1b). The reaction was similar to the preparation

of 2b using compound 4 (300 mg, 1.6 mmol) and piperonyloyl

chloride (292 mg, 1.6 mmol) to yield, after purification on a silica

gel column using hexanes-ethyl acetate(4:1), 1b (326 mg, 65%) as

yellow crystals, mp 134-136 ꢀC. ¹H NMR (CDCl₃): δ 1.30 (6H,

s), 1.97 (3H, s), 3.72 (1H, brs, NH), 5.36 (1H, s), 6.07 (2H, s), 6.45

(1H, d, J = 8.4 Hz), 6.82 (1H, d, J = 8.4 Hz), 6.90 (2H, d, J = 8.4

Hz), 7.62 (1H, s), 7.82 (1H, d, J = 8.4 Hz). ¹³C NMR (CDCl₃): δ

18.5, 31.0, 52.0, 101.9, 108.1, 109.9, 113.0, 116.7, 121.1, 122.2,

123.9, 126.0, 128.1, 129.3, 141.1, 142.2, 147.8, 152.0, 165.2. ESI-

MS: [M þ H]^þm/z 338, [M þ Na]^þm/z 360. HRESI-MS: [M þ

Na]^þm/z 360.1215 (calcd 360.1212). Anal. (C₂₀H₁₉NO₄) C, H, N.

Bis(1,2-dihydro-2,2,4-trimethylquinolin-6-yl) Isophthalate (5).

The bis(1,2-dihydro-2,2,4-trimethylquinolin-6-yl) isophthalate

is a dimer of 4 and was prepared under similar conditions to

those employed to synthesize 1a,b using compound 4 (300 mg,

1.6 mmol) and isophthaloyl dichloride (161 mg, 0.8 mmol) to

yield, after purification on a silica gel column using hexa-

nes-ethyl acetate (4:1), 5 (282 mg, 70%) as yellow crystals,

mp 213-215 ꢀC. ¹H NMR (CDCl₃): δ 1.31 (12H, s), 1.98 (6H, s),

3.70 (2H, brs, NH), 5.38 (2H, s), 6.50 (2H, d, J=8.4 Hz), 6.87

(2H, d, J = 8.4 Hz), 6.93 (2H, s), 7.66-7.68 (1H, m), 8.44 (2H, d,

J=7.5 Hz), 9.00 (1H, s). ¹³C NMR (CDCl₃): δ 18.5, 31.0, 52.1,

113.1, 116.6, 121.0, 122.4, 128.1, 128.9, 129.3, 130.6, 131.6,

134.7, 141.1, 142.1, 165.0. ESI-MS: [M þ H]^þm/z 509, [M þ

Na]^þm/z 531. HRESI-MS: [M þ H]^þm/z 509.2454 (calcd

509.2440). Anal. (C₃₂H₃₂N₂O₄) calcd: C, 75.57; H, 6.34; N,

5.51. Found: C, 74.97; H, 6.31; N, 5.40.

Quinolin-6-yl 3,5-Dimethoxybenzoate (2a). A mixture of

6-hydroxyquinoline (300 mg, 2.07 mmol) and 3,5-dimethoxy-

benzoyl chloride (414 mg, 2.07 mmol) in Et₃N (10 mL) and THF

(15 mL) was allowed to stir overnight at room temperature.

Excess Et₃N and solvent were evaporated under reduced pres-

sure, and the residue was further purified on a silica gel column

using hexanes-ethyl acetate (6:4) to yield 2a (383 mg, 60%) as

1

a white powder, mp 102-104 ꢀC. H NMR (CDCl₃): δ 3.88

(6H, s), 6.75 (1H, s), 7.38 (2H, 2s), 7.43 (1H, m), 7.59 (1H, dd, J₁

= 9.2 and J₂= 2.2 Hz), 7.69 (1H, d, J = 2.2 Hz), 8.17 (2H, m),

8.93 (1H, d, J = 3.3 Hz). ¹³C NMR (CDCl₃): δ 55.7, 106.6,

107.8, 118.6, 121.6, 124.8, 128.6, 131.1, 131.1, 135.8, 146.4,

148.8, 150.3, 160.9, 164.9. ESI-MS: [M þ H]^þm/z 310, [M þ

Na]^þm/z 332. HRESI-MS: [M þ H]^þm/z 310.1079 (calcd

310.1079). Anal. (C₁₈H₁₅NO₄) C, H,N.

976 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3

Fotie et al.

Preparation of 6-O-Ether Derivatives. These compounds were

prepared by allowing 4, an appropriate benzyl halide (1.5 equiv),

and NaH (1.5 equiv) in DMF to stir for 48 h at room tempera-

ture under a nitrogen atmosphere.¹⁰

3-(trifluoromethyl)benzyl bromide (379 mg, 1.6 mmol) were

used under the conditions described above to yield, after

purification on a silica gel column using hexanes-ethyl acet-

1

ate (9:1), 6f (259 mg, 47%) as a brown sticky oil. H NMR

6-(Benzyloxy)-1,2-dihydro-2,2,4-trimethylquinoline (6a). Com-

pound 4 (300 mg, 1.6 mmol) and benzyl bromide (271 mg, 1.6

mmol) were reacted under the conditions described above to yield,

after purification on a silica gel column using hexanes-ethyl

acetate (9:1), 6a (257 mg, 58%) as a brown sticky oil. ¹H NMR

(DMSO-d₆): δ 1.16 (6H, s), 1.87 (3H, s), 4.96 (2H, s), 5.31 (1H, s),

5.43 (1H, brs, NH) (based on HSQC data), 6.41 (1H, d, J = 8.1

Hz), 6.62-6.64 (2H, m), 7.30-7.44 (5H, m). ¹³C NMR (DMSO-

d₆): δ 18.2, 30.2, 51.0, 70.0, 110.5, 112.9, 115.0, 121.3, 127.5, 127.6,

128.3, 129.5, 137.8, 138.6, 149.5. ESI-MS: [M þ H]^þm/z 280.

HRESI-MS: [M þ H]^þm/z 280.1715 (calcd 280.1701). Anal. (C₁₉-

H₂₁NO) C, H, N.

6-(3,5-Dimethoxybenzyloxy)-1,2-dihydro-2,2,4-trimethylquino-

line (6b). Compound 4 (300 mg, 1.6 mmol) and 3,5-dimethox-

ybenzyl bromide (366 mg, 1.6 mmol) were used under the

conditions described above to yield, after purification on a silica

gel column using hexanes-ethyl acetate (9:1), 6b (307 mg, 57%)

as a brown sticky oil. ¹H NMR (DMSO-d₆): δ 1.16 (6H, s), 1.87

(3H, s), 3.72 (6H, s), 4.90 (2H, s), 5.30 (1H, s), 5.42 (1H, brs,

NH), 6.39-6.42 (2H, m), 6.58- 6.63 (4H, m). ¹³C NMR

(DMSO-d₆): δ 18.7, 30.7, 51.5, 55.6, 70.3, 99.7, 105.7, 111.0,

113.4, 115.5, 121.7, 128.0, 130.0, 139.1, 140.8, 149.9, 160.9. ESI-

MS: [M þ H]^þm/z 340. HRESI-MS: [M þ H]^þm/z 340.1898

(calcd 340.1912). Anal. (C₂₁H₂₅NO₃) C, H, N.

6-(4-Methoxybenzyloxy)-1,2-dihydro-2,2,4-trimethylquinoline

(6c). Compound 4 (300 mg, 1.6 mmol) and 4-methoxybenzyl

bromide (319 mg, 1.6 mmol) were used under the conditions

described above to yield, after purification on a silica gel column

using hexanes-ethyl acetate (9:1), 6c (245 mg, 50%) as a brown

sticky oil. ¹H NMR (DMSO-d₆): δ 1.16 (6H, s), 1.87 (3H, s), 3.74

(3H, s), 4.87 (2H, s), 5.30 (1H, s), 5.40 (1H, brs, NH), 6.41 (1H, d,

J = 8.1 Hz), 6.60-6.63 (2H, m), 6.92 (2H, d, J = 8.5 Hz), 7.34

(2H, d, J = 8.5 Hz). ¹³C NMR (DMSO-d₆): δ 18.7, 30.7, 51.5,

55.5, 70.2, 111.0, 113.4, 114.2, 115.5, 121.8, 128.0, 129.8, 130.0,

130.2, 139.1, 150.0, 159.3. ESI-MS: [M þ H]^þm/z 310. HRESI-

MS: [M þ H]^þm/z 310.1812 (calcd 310.1807). Anal. (C₂₀H₂₃-

NO₂) C, H, N.

6-(2-Methoxybenzyloxy)-1,2-dihydro-2,2,4-trimethylquino-

line (6d). Compound 4 (300 mg, 1.6 mmol) and 2-methoxy-

benzyl chloride (248 mg, 1.6 mmol) were used under the

conditions described above to yield, after purification on a

silica gel column using hexanes-ethyl acetate (9:1), 6d (281.1

mg, 57%) as a brown sticky oil. ¹H NMR (DMSO-d₆): δ 1.16

(6H, s), 1.87 (3H, s), 3.81 (3H, s), 4.93 (2H, s), 5.30 (1H, s), 5.42

(1H, brs, NH), 6.41 (1H, d, J = 8.1 Hz), 6.60-6.63 (2H, m),

6.95-6.97 (1H, m), 7.02 (1H, d, J = 8.1 Hz), 7.30-7.35 (1H,

m), 7.39 (1H, d, J = 7.5 Hz). ¹³C NMR (DMSO-d₆): δ 18.7,

30.7, 51.5, 55.8, 65.5, 110.8, 111.2, 113.4, 115.3, 120.7, 121.8,

126.0, 128.0, 129.4, 129.5, 130.0, 139.1, 150.2, 157.2. ESI-MS:

[M þ H]^þm/z 310. HRESI-MS: [M þ H]^þm/z 310.1803 (calcd

310.1807). Anal. (C₂₀H₂₃NO₂) C, H, N.

(DMSO-d₆): δ 1.16 (6H, s), 1.87 (3H, s), 5.07 (2H, s), 5.31 (1H,

s), 5.44 (1H, brs, NH), 6.41 (1H, d, J = 8.1 Hz), 6.64-6.67 (2H,

m), 7.61-7.66 (2H, m), 7.72-7.75 (2H, m). ¹³C NMR (DMSO-

d₆): δ 18.6, 30.8, 51.5, 69.7, 111.2, 113.4, 115.7, 121.8, 124.3 (q,

³J_CF=3.8 Hz), 124.7 (q, ³J_CF=3.8 Hz), 124.8 (q, ¹J_CF=270.8

Hz), 127.9, 129.7 (q, ²J_CF=32.3 Hz), 130.0, 132.0, 139.4, 139.9,

149.7. ESI-MS: [M þ H]^þm/z 348. HRESI-MS: [M þ H]^þm/z

348.1581 (calcd 348.1575). Anal. (C₂₀H₂₀F₃NO) C, F, H, N.

6-(4-(Trifluoromethyl)benzyloxy)-1,2-dihydro-2,2,4-trimethyl-

quinoline (6g). Compound 4 (300 mg, 1.6 mmol) and 4-

(trifluoromethyl)benzyl bromide (379 mg, 1.6 mmol) were used

under the conditions described above to yield, after purification

on a silica gel column using hexanes-ethyl acetate (9:1), 6g (286

mg, 52%) as a brown sticky oil that solidified upon standing. ¹H

NMR (DMSO-d₆): δ 1.16 (6H, s), 1.87 (3H, s), 5.08 (2H, s), 5.31

(1H, s), 5.44 (1H, brs, NH), 6.40 (2H, d, J = 8.1 Hz), 6.62-6.65

(2H, m), 7.64 (2H, d, J=7.5 Hz), 7.74 (2H, d, J=7.5 Hz). ¹³

NMR (DMSO-d₆): δ 18.7, 30.8, 51.5, 69.6, 111.0, 113.3, 115.5,

C

1

3

121.8, 124.7 (q, J_CF=270.75 Hz), 125.7 (q, J_CF=3.75 Hz),

127.9, 128.4 (q, ²J_CF=31.50 Hz), 130.0, 139.3, 143.3, 149.6. ESI-

MS: [MþH]^þm/z 348. HRESI-MS: [M þ H]^þm/z 348.1573

(calcd 348.1575). Anal. (C₂₀H₂₀F₃NO) C, F, H, N.

1,2-Dihydro-2,2,4-trimethyl-6-(phenethyloxy)quinoline (7).

Compound 4 (300 mg, 1.6 mmol) and (2-bromoethyl)benzene

(293 mg, 1.6 mmol) were used under the conditions described

above to yield, after purification on a silica gel column using

hexanes-ethyl acetate (9:1), 7 (93 mg, 20%) as a brown sticky

oil. ¹H NMR (DMSO-d₆): δ 1.16 (6H, s), 1.87 (3H, s), 2.97 (2H,

t, J = 6.9 Hz), 4.05 (2H, t, J = 6.9 Hz), 5.30 (1H, s), 5.39 (1H,

brs, NH), 6.40 (1H, d, J=8.1 Hz), 6.54-6.57 (2H, m), 7.20-7.26

(5H, m). ¹³C NMR (DMSO-d₆): δ 18.7, 30.7, 35.8, 51.4, 69.3,

110.6, 113.4, 115.2, 121.8, 126.6, 128.0, 128.7, 129.4, 130.0,

139.0, 139.1, 150.0. ESI-MS: [M þ H]^þm/z 294. HRESI-MS:

[M þ H]^þm/z 294.1859 (calcd 294.1858). Anal. (C₂₀H₂₃NO)

C, H, N.

1-(3-Methoxybenzyl)-6-(3-methoxybenzyloxy)-1,2-dihydro-

2,2,4-trimethylquinoline (8e). 8e, a side product from the pre-

paration of 6e, was obtained as a yellow sticky oil (157 mg,

1

23%). H NMR (DMSO-d₆): δ 1.28 (6H, s), 1.94 (3H, s), 3.70

(3H, s), 3.73 (3H, s), 4.37 (2H, s), 4.91 (2H, s), 5.46 (1H, s), 6.11

(1H, d, J = 8.7 Hz), 6.55 (1H, dd, J₁=8.7 and J₂= 2.7 Hz), 6.70

(1H, d, J = 2.7 Hz), 6.75 (1H, d, J = 8.4 Hz), 6.85-6.97 (5H, m),

7.17-7.27 (2H, m). ¹³C NMR (DMSO-d₆): δ 18.8, 28.0, 47.9,

55.3, 55.5, 56.9, 70.0, 111.5, 111.7, 112.6, 112.7, 113.5, 113.6,

114.4, 118.7, 120.1, 124.1, 127.4, 129.9, 130.0, 131.4, 138.6,

139.8, 142.4, 150.1, 159.8, 159.9. ESI-MS: [M þ H]^þm/z 430,

[M þ Na]^þm/z 452. HRESI-MS: [M þ Na]^þm/z 452.2204 (calcd

452.2202). Anal. (C₂₈H₃₁NO₃) C, H, N.

1-(3-(Trifluoromethyl)benzyl)-6-(3-(trifluoromethyl)benzyloxy)-

1,2-dihydro-2,2,4-trimethylquinoline (8f). 8f, a side product from

the preparation of 6f, was obtained as yellow sticky oil that

1

6-(3-Methoxybenzyloxy)-1,2-dihydro-2,2,4-trimethylquinoline

(6e). Compound 4 (300 mg, 1.6 mmol) and 3-methoxybenzyl

bromide (319 mg, 1.6 mmol) were used under the conditions

described above to yield, after purification on a silica gel column

using hexanes-ethyl acetate (9:1), 6e (267.8 mg, 55%) as a

solidified upon standing (280 mg, 35%). H NMR (DMSO-d₆):

δ 1.30 (6H, s), 1.94 (3H, s), 4.52 (2H, s), 5.04 (2H, s), 5.48 (1H, s),

6.10 (1H, d, J=8.7 Hz), 6.58 (1H, dd, J₁=8.7 and J₂=2.1 Hz), 6.75

(1H, d, J = 2.1 Hz), 7.50-7.69 (8H, m). ¹³C NMR (DMSO-d₆): δ

18.7, 27.8, 47.5, 57.0, 69.3, 111.8, 112.5, 114.5, 123.0 (q, ³J_CF=3.75

Hz), 123.6 (q, ³J_CF=3.75 Hz), 123.7 (q, ³J_CF=3.75 Hz), 124.3 (q,

³J_CF=3.75 Hz), 124.8 (q, ¹J_CF= 271.2 Hz), 125.8 (q, ¹J_CF= 272.0

Hz), 127.4, 129.6 (q, ²J_CF= 36.0 Hz), 131.6 (q, ²J_CF=27.75 Hz),

138.5, 139.7, 142.4, 150.1. ESI-MS: [M þ H]^þm/z 506, [M þ Na]^þ

m/z 528. HRESI-MS: [M þ H]^þm/z 506.1904 (calcd 506.1918).

Anal. (C₂₈H₂₅F₆NO) C, F, H, N.

1

brown sticky oil. H NMR (DMSO-d₆): δ 1.17 (6H, s), 1.88

(3H, s), 3.76 (3H, s), 4.94 (2H, s), 5.31 (1H, s), 5.42 (1H, brs,

NH), 6.41 (1H, d, J = 8.1 Hz), 6.62-6.65 (2H, m), 6.88 (1H, d,

J = 8.4 Hz), 6.99-7.01 (2H, m), 7.26-7.32 (1H, m). ¹³C NMR

(DMSO-d₆): δ 18.7, 30.7, 51.5, 55.5, 70.3, 111.0, 113.4, 113.5,

113.6, 115.5, 120.1, 121,8, 128.0, 129.9, 130.0, 139.2, 140.0,

150.0, 159.8. ESI-MS: [M þ H]^þm/z 310. HRESI-MS: [M þ

H]^þm/z 310.1805 (calcd 310.1807). Anal. (C₂₀H₂₃NO₂) C, H, N.

6-(3-(Trifluoromethyl)benzyloxy)-1,2-dihydro-2,2,4-trimethyl-

1-(4-(Trifluoromethyl)benzyl)-6-(4-(trifluoromethyl)benzyloxy)-

1,2-dihydro-2,2,4-trimethylquinoline (8g). 8g, a side product from

the preparation of 6g, was obtained as a yellow sticky oil (257 mg,

1

quinoline (6f). Compound

4

(300 mg, 1.6 mmol) and

32%). H NMR (DMSO-d₆): δ 1.29 (6H, s), 1.95 (3H, s), 4.51

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 977

(2H, s), 5.05 (2H, s), 5.49 (1H, s), 6.06 (1H, d, J = 8.7 Hz), 6.56

(1H, d, J = 8.7 Hz), 6.74 (1H, s), 7.52 (2H, d, J = 8.1 Hz), 7.61

(2H, d, J = 9.0 Hz), 7.64 (2H, d, J = 9.0 Hz), 7.71 (2H, d, J = 8.1

Hz). ¹³C NMR (DMSO-d₆): δ 18.7, 27.9, 47.5, 57.0, 69.2, 111.8,

ybenzyl bromide (916 mg, 3.96 mmol) were reacted under the

conditions described above to yield, after purification on a silica

gel column using hexanes-DCM (1:1), 1-(3,5-dimethoxybenzyl)-

1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9b) (762 mg, 85%) as a

brown oil. 9b (300 mg, 0.88 mmol) was further acetylated to yield,

after purification on a silica gel column using hexanes-DCM (1:1),

10b (310 mg, 92%) as a yellow oil that solidified upon standing. ¹H

NMR (DMSO-d₆): δ 1.31 (6H, s), 1.92 (3H, s), 2.18 (3H, s), 3.69

(6H, s), 4.40(2H, s), 5.47(1H, s), 6.15 (1H, d, J = 8.7 Hz), 6.34 (1H,

d, J=2.5Hz),6.46(2H, m), 6.62(1H, dd,J₁=8.7 and J₂=2.7Hz),

6.72 (1H, d, J = 2.7 Hz). ¹³C NMR (DMSO-d₆): δ 18.7, 21.2, 28.5,

48.0, 57.3, 60.2, 98.1, 104.6, 112.0, 116.9, 121.4, 123.3, 126.9, 131.1,

141.4, 141.9, 142.8, 161.1, 170.1. ESI-MS: [M þ Na]^þm/z 404.

HRESI-MS: [M þ Na]^þm/z 404.1832 (calcd 404.1838). Anal.

(C₂₃H₂₇NO₄) C, H, N.

1-(4-Cyanobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10c). Compound 4 (500 mg, 2.6 mmol) and 4-

(bromomethyl)benzonitrile (784 mg, 4.0 mmol) were reacted

under the conditions described above to yield, after purifica-

tion on a silica gel column using hexanes-ethyl acetate (8:2),

4-((6-hydroxy-2,2,4-trimethylquinolin-1(2H)-yl)methyl)benzoni-

trile (9c) (684 mg, 86%). This latter compound was further

acetylated to yield, after purification on a silica gel column

using hexanes-ethyl acetate (9:1), 10c (739 mg, 94.9%) as a

yellow oil that solidified upon standing. ¹H NMR (DMSO-d₆):

δ 1.31 (6H, s), 1.92 (3H, s), 2.18 (3H, s), 4.59 (2H, s), 5.50 (1H,

s), 6.07 (1H, d, J=8.7 Hz), 6.53 (1H, dd, J₁=8.7 Hz and J₂=2.4

Hz), 6.74 (1H, d, J = 2.4 Hz), 7.50 (2H, d, J=8.1 Hz), 7.77 (2H,

d, J=8.1 Hz). ¹³C NMR (DMSO d₆): δ 18.7, 21.2, 28.4, 47.6,

57.4, 109.8, 111.8, 117.2, 119.4, 121.5, 123.6, 126.9, 127.6,

131.2, 132.9, 141.5, 141.6, 146.5, 170.1. ESI-MS: [M þ H]^þm/z

347. HRESI-MS: [M þ H]^þm/z 347.1752 (calcd 347.1760).

Anal. (C₂₂H₂₂N₂O₂) C, H, N.

1-(3-Cyanobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl Acet-

ate (10d). Compound 4 (300 mg, 1.6 mmol) and 3-(bromo-

methyl)benzonitrile (470 mg, 2.4 mmol) were reacted under the

conditions described above to yield, after purification on a silica gel

column using hexanes-ethyl acetate (8:2), 3-((6-hydroxy-2,2,4-

trimethylquinolin-1(2H)-yl)methyl)benzonitrile (9d) (386 mg,

79.3%). This latter compound was further acetylated to yield, after

purification on a silica gel column using hexanes-ethyl acetate

(9:1), 10d (382 mg, 87.0%) as a yellow oil that solidified upon

standing. ¹H NMR (CDCl₃): δ 1.35 (6H, s), 2.01 (3H, s), 2.25

(3H, s), 4.48 (2H, s), 5.40 (1H, s), 6.05 (1H, d, J=8.7 Hz), 6.62

(1H, dd, J₁= 8.7 Hz and J₂=2.7 Hz), 6.81 (1H, d, J = 2.7 Hz),

7.40 (1H, m), 7.50-7.62 (3H, m). ¹³C NMR (CDCl₃): δ 18.9,

21.3, 28.5, 47.8, 57.5, 112.0, 113.0, 117.1, 119.2, 121.0, 124.3,

127.8, 129.7, 130.1, 130.5, 130.8, 131.0, 141.5, 141.6, 142.0,

170.3. ESI-MS: [M þ H]^þm/z 347. HRESI-MS: [M þ H]^þm/z

347.1758 (calcd 347.1760). Anal. (C₂₂H₂₂N₂O₂) C, H, N.

1-(4-Chlorobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl Acet-

ate (10e). Compound 4 (300 mg, 1.6 mmol) and 4-chlorobenzyl

bromide (493 mg, 2.4 mmol) were reacted under the conditions

described above to yield, after purification on a silica gel column

using hexanes-ethyl acetate (19:1), 9e (457 mg, 91%). This latter

compound was further acetylated to yield, after purification on a

silica gel column using hexanes-DCM (3:1), 10e (477 mg, 92%) as

a yellow oil that solidified upon standing. ¹H NMR (DMSO-d₆): δ

1.31 (6H, s), 1.92 (3H, s), 2.18 (3H, s), 4.48 (2H, s), 5.48 (1H, s), 6.11

(1H, d, J=8.7 Hz), 6.60 (1H, dd, J₁=8.7 Hz and J₂= 2.3 Hz), 6.73

(1H, d, J=2.3 Hz), 7.31-7.38 (4H, m). ¹³C NMR (DMSO d₆): δ

18.7, 21.2, 28.4, 47.2, 57.4, 111.9, 117.0, 121.5, 123.5, 126.9, 128.4,

128.9, 131.2, 131.3, 139.2, 141.5, 141.6, 170.1. ESI-MS: [M þ H]^þ

m/z 356. HRESI-MS: [M þ H]^þm/z 356.1421 (calcd 356.1417).

Anal. (C₂₁H₂₂ClNO₂) C, Cl, H, N.

112.5, 114.4, 124.4, 124.7 (q, ¹J_CF=270.75 Hz), 124.8 (q, ¹J_CF

=

270.75 Hz), 125.6 (q, ³J_CF= 3.75 Hz), 127.4 (q, ²J_CF= 30.75 Hz),

128.9 (q, ²J_CF=31.5 Hz), 131.6, 138.4, 143.1, 145.8, 150.0. ESI-

MS: [MþH]^þm/z506, [M þ Na]^þm/z528. HRESI-MS: [M þ H]^þ

m/z 506.1908 (calcd 506.1918). Anal. (C₂₈H₂₅F₆NO) C, F, H, N.

Preparation of N-Substituted Compounds. All of the N-sub-

stituted compounds were prepared by heating a mixture of 4 and

the appropriate benzyl halide (1.5 equiv) in refluxing toluene,

using Et₃N (1.5 equiv) as base. Since the N-substituted com-

pounds appeared to be very susceptible to auto-oxidation, they

were further protected by either acetylation or benzoylation

except for 11a, 11b, and 20a, which were obtained as needles and

thus were stable as the free hydroxy compounds.

1,2-Dihydro-2,2,4-trimethyl-1-((pyridin-2-yl)methyl)quinolin-

4 (300 mg, 1.6 mmol) and 2-

6-ol (11a). Compound

(bromomethyl)pyridine hydrobromide (401 mg, 1.6 mmol) were

reacted under the conditions described above to yield, after

purification on a silica gel column using hexanes-ethyl acetate

(7:3), 11a as brown needles (132 mg, 23%), mp 193-195 ꢀC. ¹H

NMR (DMSO-d₆): δ 1.29 (6H, s), 1.91 (3H, s), 4.41 (2H, s), 5.45

(1H, s), 6.00 (1H, d, J = 8.7 Hz), 6.30 (1H, dd, J₁= 8.7 and J₂=

2.7 Hz), 6.52 (1H, d, J = 2.7 Hz), 7.18-7.22 (1H, m), 7.27 (1H,

d, J = 7.8 Hz), 7.62-7.68 (1H, m), 8.50 (1H, brs, OH), 8.52 (1H,

d, J=4.5 Hz). ¹³C NMR (DMSO): δ 18.8, 27.7, 50.5, 56.8, 111.3,

112.7, 114.9, 120.8, 122.3, 124.4, 127.5, 131.4, 137.0, 137.2,

148.8, 149.4, 161.0. The HSQC and HMBC data enabled us to

find the key correlation between the protons at δ 4.41 ppm

(benzylic protons) and the quaternary carbon (C-2) at δ 56.8

ppm. The H-3 proton (5.45 ppm) also displayed a correlation

with the carbon at δ 56.8 ppm, confirming the identity of this

carbon as C-2. These data demonstrate that the methylpyridinyl

side chain is bound to the N1-atom. ESI-MS: [M þ H]^þm/z 281.

HRESI-MS: [M þ H]^þm/z 281.1659 (calcd 281.1654). Anal.

(C₁₈H₂₀N₂O) C, F, H, N.

1,2-Dihydro-2,2,4-trimethyl-1-((pyridin-4-yl)methyl)quinolin-

6-ol (11b). Compound

4 (300 mg, 1.6 mmol) and 4-

(bromomethyl)pyridine hydrobromide (401 mg, 1.6 mmol) were

reacted under the conditions described above to yield, after

purification on a silica gel column using hexanes-ethyl acetate

(7:3), 11b as brown needles (162 mg, 36%), mp 187-188 ꢀC. ¹H

NMR (DMSO-d₆): δ 1.26 (6H, s), 1.92 (3H, s), 4.40 (2H, s), 5.45

(1H, s), 5.95 (1H, d, J = 8.7 Hz), 6.31 (1H, dd, J₁=8.7 and J₂=

2.4 Hz), 6.53 (1H, d, J = 2.4 Hz), 7.30 (2H, d, J = 5.4 Hz), 8.45

(2H, d, J = 5.4 Hz), 8.51 (1H, brs, OH). ¹³C NMR (DMSO-d₆):

δ 18.8, 27.6, 47.1, 56.8, 111.4, 112.7, 114.9, 122.2, 124.3, 127.5,

131.3, 136.7, 148.9, 150.0, 150.6. ESI-MS: [M þ H]^þm/z 281.

HRESI-MS: [M þ H]^þm/z 281.1654 (calcd 281.1654). Anal.

(C₁₈H₂₀N₂O) calcd: C, 77.11; H, 7.19; N, 9.99. Found: C, 76.63;

H, 7.20; N, 9.83.

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl Acetate(10a).

Compound 4 (300 mg, 1.6mmol) and benzyl bromide (410 mg, 2.4

mmol) were reacted under the conditions described above to yield,

after purification on silica gel column using hexanes-ethyl acetate

(9:1), 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9a) (398

mg, 89%). 9a (300 mg, 1.07 mmol) was further acetylated to yield,

after purification on a silica gel column using hexanes-DCM

(1:1), 10a (328 mg, 95%) as yellow oil that solidified upon

1

standing. H NMR (DMSO-d₆): δ 1.33 (6H, s), 1.92 (3H, s),

2.17 (3H, s), 4.89 (2H, s), 5.49 (1H, s), 6.14 (1H, d, J = 8.7 Hz),

6.58 (1H, dd, J₁=8.7 and J₂= 2.7 Hz), 6.73 (1H, d, J = 2.7 Hz),

7.18-7.20 (5H, m). ¹³C NMR(DMSO-d₆):δ18.7, 21.2, 28.5, 47.8,

57.3, 112.0, 116.9, 121.4, 123.4, 126.5, 127.0, 128.9, 131.2, 140.1,

141.3, 141.9, 170.1. ESI-MS: [M þ Na]^þm/z 344. HRESI-MS: [M þ

Na]^þm/z 344.1624 (calcd 344.1626). Anal. (C₂₁H₂₃NO₂) C, H, N.

1-(3,5-Dimethoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10b). Compound 4(500 mg, 2.64 mmol) and 3,5-dimethox-

1-(4-Methylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10f). Compound 4 (300 mg, 1.6 mmol) and 4-me-

thylbenzyl bromide (444 mg, 2.4 mmol) were reacted

under the conditions described above to yield, after purification

on a silica gel column using hexanes-ethyl acetate (9:1),

978 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3

Fotie et al.

1-(4-methylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9f)

(400 mg, 85%). This latter compound was further acetylated to

yield, after purification on a silica gel column using hexanes-DCM

(3:1), 10f (434 mg, 95%) as a yellow oil. ¹H NMR (DMSO-d₆): δ

1.32 (6H, s), 1.91 (3H, s), 2.19 (3H, s), 2.22 (3H, s), 4.43 (2H, s), 5.47

(1H, s), 6.13(1H, d, J= 8.7Hz), 6.58(1H, dd, J₁=8.7 Hz and J₂=

2.7 Hz), 6.72 (1H, d, J=2.7 Hz), 7.01 (2H, d, J = 7.8 Hz), 7.20 (2H,

d, J = 7.8 Hz). ¹³C NMR (DMSO d₆): δ 18.7, 21.1, 21.2, 28.5, 47.6,

57.3, 112.0, 116.9, 121.4, 123.3, 126.4, 126.9, 129.5, 131.2, 135.9,

136.9, 141.3, 141.9, 170.1. ESI-MS: [M þ H]^þm/z 336. HRESI-

MS: [M þ H]^þm/z 336.1966 (calcd 336.1964). Anal. (C₂₂H₂₅NO₂)

C, H, N.

acetylated to yield, after purification on a silica gel column using

hexanes-DCM (7:3), 10j (363 mg, 70%) as a yellow oil that

solidified upon standing. ¹H NMR (CDCl₃): δ 1.37 (6H, s), 2.02

(3H, s), 2.26 (3H, s), 4.54 (2H, s), 5.40 (1H, s), 6.10 (1H, d, J = 8.7

Hz), 6.63(1H, dd, J₁=8.7 Hz and J₂= 2.7 Hz), 6.81 (1H, d, J=2.7

Hz), 7.46 (2H, d, J = 8.1 Hz), 7.57 (2H, d, J = 8.1 Hz). ¹³C NMR

(CDCl₃): δ 18.7, 21.1, 28.5, 47.9, 57.3, 111.7, 116.7, 120.8, 123.8,

124.8 (q, ¹J_CF=270.75 Hz), 125.7 (q, ³J_CF=3.75 Hz), 126.5, 127.4,

129.1 (q, ²J_CF=32.25 Hz), 130.3, 141.5, 143.8, 170.2. ESI-MS: [M þ

H]^þm/z390. HRESI-MS: [MþH]^þm/z390.1682 (calcd 390.1681).

Anal. (C₂₂H₂₂F₃NO₂) C, F, H, N.

1-(3-Methylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10k). Compound 4 (300 mg, 1.6 mmol) and 3-methyl-

benzyl bromide (444 mg, 2.4 mmol) were reacted under the

conditions described above to yield, after purification on a

silica gel column using hexanes-ethyl acetate (9:1), 1-(3-

methylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9k)

(415 mg, 88%). This latter compound was further acetylated

to yield, after purification on a silica gel column using hex-

anes-DCM (7:3), 10k (425 mg, 90%) as a yellow oil. ¹H NMR

(CDCl₃): δ 1.38 (6H, s), 2.02 (3H, s), 2.27 (3H, s), 2.37 (3H, s),

4.47 (2H, s), 5.39 (1H, s), 6.22 (1H, d, J = 8.7 Hz), 6.63 (1H, dd,

J₁=8.7 Hz and J₂= 2.4 Hz), 6.79 (1H, d, J = 2.4 Hz), 7.05 (1H,

m), 7.17-7.21 (3H, m). ¹³C NMR (CDCl₃): δ 18.7, 21.1, 21.5,

28.6, 48.3, 57.2, 112.0, 116.4, 120.8, 123.2, 123.5, 126.7, 127.4,

127.5, 128.5, 130.3, 138.2, 139.4, 141.2, 142.1, 170.3. ESI-MS:

[M þ H]^þm/z 336. HRESI-MS: [M þ H]^þm/z 336.1964 (calcd

336.1964). Anal. (C₂₂H₂₅NO₂) C, H, N.

1-(3-Methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10l). Compound 4 (300 mg, 1.6 mmol) and 3-methox-

ybenzyl bromide (483 mg, 2.4 mmol) were reacted under the

conditions described above to yield, after purification on a silica

gel column using hexanes-ethyl acetate (9:1), 1-(3-meth-

oxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9l) (432

mg, 88%). This latter compound was further acetylated to yield,

after purification on a silica gel column using hexanes-DCM

(7:3), 10l (383 mg, 78%) as a yellow oil. ¹H NMR (CDCl₃): δ 1.38

(6H, s), 2.00 (3H, s), 2.26 (3H, s), 3.79 (3H, s), 4.46 (2H, s), 5.37

(1H, s), 6.21 (1H, d, J = 8.7 Hz), 6.62 (1H, dd, J₁= 8.7 Hz and

J₂= 2.7 Hz), 6.77 (1H, d, J = 2.7 Hz), 6.92-6.97 (2H, m),

7.21-7.28 (2H, m). ¹³C NMR (CDCl₃): δ 18.7, 21.1, 28.5, 48.2,

55.2, 57.2, 111.6, 111.9, 112.1, 116.4, 118.5, 120.7, 123.6, 127.4,

129.6, 130.2, 141.2, 141.4, 142.0, 159.9, 170.3. ESI-MS: [M þ

H]^þm/z 352. HRESI-MS: [M þ H]^þm/z 352.1913 (calcd

352.1913). Anal. (C₂₂H₂₅NO₃) C, H, N.

1-(3,4-Dichlorobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10g). Compound 4 (300 mg, 1.6 mmol) and 3,4-dichlor-

obenzyl chloride (469 mg, 2.4 mmol) were reacted under the

conditions described above to yield, after purification on a silica

gel column using hexanes-ethyl acetate (9:1), 1-(3,4-dichlo-

robenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9g) (318 mg,

57%). This latter compound was further acetylated to yield, after

purification on a silica gel column using hexanes-DCM (3:1), 10g

1

(346 mg, 97%) as a yellow oil that solidified upon standing. H

NMR (DMSO-d₆): δ 1.30 (6H, s), 1.92 (3H, s), 2.18 (3H, s), 4.50

(2H, s), 5.49 (1H, s), 6.12 (1H, d, J=8.7 Hz), 6.62 (1H, dd, J₁= 8.7

Hz and J₂= 2.4Hz), 6.75 (1H, d, J = 2.4 Hz), 7.28 (1H, d, J = 8.1

Hz), 7.52-7.57 (2H, m). ¹³C NMR (DMSO d₆): δ 18.7, 21.2, 28.3,

46.8, 57.4, 111.9, 117.2, 121.5, 123.6, 126.9, 128.5, 129.3, 131.1,

131.3, 131.5, 141.4, 141.7, 141.8, 170.1. ESI-MS: [M þ H]^þm/z

390. HRESI-MS: [M þ H]^þm/z 390.1031 (calcd 390.1028). Anal.

(C₂₁H₂₁Cl₂NO₂) C, Cl, H, N.

1-(3-Chlorobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl Acet-

ate (10h). Compound 4 (300 mg, 1.6 mmol) and 3-chlorobenzyl

bromide (493 mg, 2.4 mmol) were reacted under the conditions

described above to yield, after purification on a silica gel column

using hexanes-ethyl acetate (9:1), 1-(3-chlorobenzyl)-1,2-dihydro-

2,2,4-trimethylquinolin-6-ol (9h) (467 mg, 93%). This latter com-

pound was further acetylated to yield, after purification on a silica

gel column using hexanes-DCM (3:1), 10h (466 mg, 88%) as a

1

yellow oil that solidified upon standing. H NMR (DMSO-d₆): δ

1.31 (6H, s), 1.92 (3H, s), 2.18 (3H, s), 4.51 (2H, s), 5.49 (1H, s), 6.14

(1H, d, J=8.7 Hz), 6.62 (1H, dd, J₁=8.7 Hz and J₂= 2.4 Hz), 6.75

(1H, d, J = 2.4 Hz), 7.25-7.34 (4H, m). ¹³C NMR (DMSO d₆): δ

18.7, 21.2, 28.4, 47.3, 57.4, 111.9, 117.1, 121.5, 123.5, 125.2, 126.3,

126.9, 127.0, 130.8, 131.2, 133.6, 141.6, 141.7, 143.1, 170.1. ESI-MS:

[M þ H]^þm/z 356. HRESI-MS: [M þ H]^þm/z 356.1420 (calcd

356.1417). Anal. (C₂₁H₂₂ClNO₂) C, Cl, H, N.

1-(3-(Trifluoromethyl)benzyl)-1,2-dihydro-2,2,4-trimethylqui-

nolin-6-yl Acetate (10i). Compound 4 (300 mg, 1.6 mmol) and

3-(trifluoromethyl)benzyl bromide (574 mg, 2.4 mmol) were

reacted under the conditions described above to yield, after

purification on a silica gel column using hexanes-ethyl acetate

(9:1), 1-(3-(trifluoromethyl)benzyl)-1,2-dihydro-2,2,4-trimethyl-

quinolin-6-ol (9i) (457 mg, 82%). This latter compound was

further acetylated to yield, after purification on a silica gel column

using hexanes-DCM (3:1), 10i (497 mg, 97%) as a yellow oil that

solidified upon standing. ¹H NMR (CDCl₃): δ 1.37 (6H, s), 2.02

(3H, s), 2.26 (3H, s), 4.53 (2H, s), 5.40 (1H, s), 6.12 (1H, d, J = 8.7

Hz), 6.63 (1H, dd, J₁= 8.7 Hz and J₂= 2.4 Hz), 6.81 (1H, d, J =

2.4 Hz), 7.41-7.60(4H, m). ¹³C NMR(CDCl₃): δ 18.7, 21.1, 28.4,

47.9, 57.3, 111.7, 116.7, 120.8, 122.9 (q, ³J_CF= 3.5 Hz), 123.7 (q,

³J_CF=3.5 Hz), 123.8, 126.0 (q, ¹J_CF= 271.8 Hz), 127.5, 129.1 (q,

²J_CF=32.3 Hz), 132.0, 140.7, 141.5, 141.6, 170.2. ESI-MS: [M þ

H]^þm/z 390. HRESI-MS: [M þ H]^þm/z 390.1682 (calcd

390.1681). Anal. (C₂₂H₂₂F₃NO₂) C, F, H, N.

1-(4-Fluorobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10m). Compound 4 (300 mg, 1.6 mmol) and 4-fluor-

obenzyl bromide (454 mg, 2.4 mmol) were reacted under the

conditions described above to yield, after purification on a silica

gel column using hexanes-ethyl acetate (9:1), 1-(4-fluoro-

benzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9m) (400 mg,

85%). This latter compound was further acetylated to yield, after

purification on a silica gel column using hexanes-DCM (3:1), 10m

1

(429 mg, 94%) as a yellow oil that solidified upon standing. H

NMR (CDCl₃): δ 1.36 (6H, s), 2.00 (3H, s), 2.26 (3H, s), 4.45 (2H,

s), 5.38 (1H, s), 6.15 (1H, d, J = 8.7 Hz), 6.62 (1H, dd, J₁= 8.7 Hz

and J₂= 2.4 Hz), 6.79 (1H, d, J = 2.4 Hz), 6.97-7.02 (2H, m),

7.28-7.32 (2H, m). ¹³C NMR (CDCl₃): δ 18.7, 21.1, 28.5, 47.5,

57.2, 111.8, 115.4 (²J_CF=21.75 Hz), 116.5, 120.7, 123.7, 127.4, 127.6

(³J_CF=8.25 Hz), 130.3, 134.9, 141.3, 141.7, 161.7 (¹J_CF=242.25

Hz), 170.2. ESI-MS: [M þ H]^þm/z 340. HRESI-MS: [M þ H]^þ

m/z 340.1714 (calcd 340.1713). Anal. (C₂₁H₂₂FNO₂) C, F, H, N.

1-(4-Methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10n). Compound 4 (300 mg, 1.6 mmol) and 4-methox-

ybenzyl bromide (483 mg, 2.4 mmol) were reacted under the

conditions described above to yield, after purification on a silica

gel column using hexanes-ethyl acetate (9:1), 1-(4-meth-

oxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9n) (441

mg, 90%). This latter compound was further acetylated to yield,

after purification on a silica gel column using hexanes-DCM

1-(4-(Trifluoromethyl)benzyl)-1,2-dihydro-2,2,4-trimethylqui-

nolin-6-yl Acetate (10j). Compound 4 (300 mg, 1.6 mmol) and

4-(trifluoromethyl)benzyl bromide (574 mg, 2.4 mmol) were

reacted under the conditions described above to yield, after

purification on a silica gel column using hexanes-ethyl acetate

(9:1), 1-(4-(trifluoromethyl)benzyl)-1,2-dihydro-2,2,4-trimethyl-

quinolin-6-ol (9j) (463 mg, 84%). This latter compound was further

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 979

(3:1), 10n (366 mg, 73%) as a yellow oil that solidified upon

standing. H NMR (CDCl₃): δ 1.37 (6H, s), 2.00 (3H, s), 2.26

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl Benzoate

(13a). 9a (500 mg, 1.8 mmol) and benzoyl chloride (379 mg,

2.7 mmol) were allowed to react under conditions similar to

those used in the preparation of 1a,b to yield, after purification

on a silica gel column using hexanes-DCM (8:2), 13a (570 mg,

82.6%) as a yellow oil that solidified upon standing. ¹H NMR

(CDCl₃): δ 1.42 (6H, s), 2.05 (3H, s), 4.56 (2H, s), 5.43 (1H, s),

6.29 (1H, d, J=8.7 Hz), 6.79 (1H, dd, J₁= 8.7 Hz and J₂= 2.4

Hz), 6.96 (1H, d, J=2.4 Hz), 7.26-7.30 (2H, m), 7.32-7.41 (3H,

m), 7.50-7.55 (2H, m), 7.62-7.64 (1H, m), 8.22 (2H, d, J = 7.5

Hz). ¹³C NMR (CDCl₃): δ 18.8, 28.6, 48.2, 57.3, 112.1, 116.6,

120.9, 123.7, 126.2, 126.7, 127.5, 128.5, 128.6, 128.8, 130.1,

130.3, 133.3, 139.4, 141.4, 142.1, 165.8. ESI-MS: [M þ Na]^þ

m/z 406. HRESI-MS: [M þ Na]^þm/z 406.1750 (calcd 406.1783).

Anal. (C₂₆H₂₅NO₂) C, H, N.

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl-2-phenylace-

tate (13b). 9a (700 mg, 2.5 mmol) and 2-phenylacetyl chloride

(587 mg, 3.8 mmol) were allowed to react under conditions

similar to those used in the preparation of 1a,b to yield, after

purification on a silica gel column using hexanes-DCM (85:15),

13b (847 mg, 85.2%) as a yellow oil. ¹H NMR (DMSO-d₆): δ 1.32

(6H, s), 1.92 (3H, s), 3.87 (2H, s), 4.49 (2H, s), 5.48 (1H, s), 6.15

(1H, d, J=8.7 Hz), 6.58 (1H, dd, J₁= 8.7 Hz and J₂= 2.7 Hz),

6.74 (1H, d, J=2.7 Hz), 7.18-7.35 (10H, m). ¹³C NMR (CDCl₃):

δ 18.7, 28.5, 41.4, 48.1, 57.2, 111.9, 116.3, 120.6, 123.5, 126.2,

126.6, 127.0, 127.4, 128.6, 128.7, 129.3, 130.3, 133.8, 139.4,

141.3, 142.0, 170.7. ESI-MS: [M þ Na]^þm/z 420. HRESI-MS:

[M þ Na]^þm/z 420.1925 (calcd 420.1939). Anal. (C₂₇H₂₇NO₂) C,

H, N.

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl Pivalate (13c).

9a (400 mg, 1.4 mmol) and trimethylacetyl chloride (253 mg, 2.1

mmol) were allowed to react under conditions similar to those used

in the preparation of 1a,b to yield, after purification on a silica gel

column using hexanes-DCM (8:2), 13c (464 mg, 91.2%) as a

yellow oil that solidified upon standing. ¹H NMR (DMSO-d₆): δ

1.24 (9H, s), 1.32 (6H, s), 1.93 (3H, s), 4.49 (2H, s), 5.48 (1H, s), 6.15

(1H, J = 8.7 Hz), 6.54 (1H, dd, J₁=8.7 and J₂= 2.4 Hz), 6.68 (1H,

d, J = 2.4 Hz), 7.19-7.30 (5H, m). ¹³C NMR (DMSO-d₆): δ 18.7,

27.3, 28.4, 40.9, 47.8, 57.3, 112.1, 116.7, 121.2, 123.5, 126.5, 126.9,

127.0, 128.9, 131.2, 140.1, 141.6, 141.8, 177.2. ESI-MS: [M þ Na]^þ

m/z 386. HRESI-MS: [M þ Na]^þm/z 386.2065 (calcd 386.2096).

Anal. (C₂₄H₂₉NO₂) C, H, N.

1

(3H, s), 3.80 (3H, s), 4.44 (2H, s), 5.37 (1H, s), 6.21 (1H, d, J =

8.7 Hz), 6.63 (1H, dd, J₁=8.7 Hz and J₂= 2.4 Hz), 6.78 (1H, d,

J = 2.4 Hz), 6.86 (2H, d, J=8.4 Hz), 7.26 (2H, d, J = 8.4 Hz).

¹³C NMR (CDCl₃): δ 18.7, 21.1, 28.6, 47.6, 55.3, 57.2, 111.9,

114.0, 116.4, 120.7, 123.5, 127.2, 127.3, 130.3, 131.2, 141.1,

142.0, 158.4, 170.3. ESI-MS: [M þ H]^þm/z 352. HRESI-MS:

[M þ H]^þm/z 352.1913 (calcd 352.1913). Anal. (C₂₂H₂₅NO₃) C,

H, N.

1-(4-tert-Butylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl

Acetate (10o). Compound 4 (300 mg, 1.6 mmol) and 4-(tert-

butyl)benzyl bromide (545 mg, 2.4 mmol) were reacted under the

conditions described above to yield, after purification on a silica

gel column using hexanes-ethyl acetate (9:1), 1-(4-tert-

butylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (9o) (425

mg, 80%). This latter compound was further acetylated to yield,

after purification on a silica gel column using hexanes-DCM

1

(3:1), 10o (383 mg, 80%) as a yellow oil. H NMR (CDCl₃): δ

1.33 (9H, s), 1.38 (6H, s), 2.01 (3H, s), 2.26 (3H, s), 4.47 (2H, s),

5.38 (1H, s), 6.23 (1H, d, J=8.7 Hz), 6.63 (1H, dd, J₁=8.7 Hz and

J₂= 2.4 Hz), 6.78 (1H, d, J = 2.4 Hz), 7.27 (2H, d, J=8.1 Hz),

7.34 (2H, d, J=8.1 Hz). ¹³C NMR (CDCl₃): δ 18.7, 21.1, 28.6,

31.4, 34.4, 47.9, 57.1, 112.0, 116.3, 120.7, 123.5, 125.4, 125.8,

127.4, 130.3, 136.3, 141.1, 142.1, 149.4, 170.3. ESI-MS: [M þ H]^þ

m/z 378. HRESI-MS: [M þ H]^þm/z 378.2432 (calcd 378.2433).

Anal. (C₂₅H₃₁NO₂) C, H, N.

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl-3,5-dimethoxy-

benzoate (12a). 9a (700 mg, 2.5 mmol) and 3,5-dimethoxybenzoyl

chloride (762 mg, 3.8 mmol) were allowed to react under conditions

similar to those used in the preparation of 1a,b to yield, after

purification on a silica gel column using hexanes-DCM (8:2), 12a

1

(862 mg, 78%) as a yellow oil that solidified upon standing. H

NMR (DMSO-d₆): δ 1.35 (6H, s), 1.94 (3H, s), 3.81 (6H, s), 4.52

(2H, s), 5.51 (1H, s), 6.20 (1H, d, J = 8.7 Hz), 6.74 (1H, d, J = 8.7

Hz), 6.82-6.88 (2H, m), 7.07-7.22 (2H, m), 7.28-7.33 (5H, m).

¹³C NMR (DMSO-d₆): δ 18.7, 28.5, 47.8, 56.0, 57.4, 106.2, 107.7,

112.1, 116.9, 121.4, 123.5, 126.5, 126.9, 127.0, 128.9, 131.3, 131.7,

140.0, 141.4, 142.1, 161.0, 165.2. ESI-MS: [M þ Na]^þm/z 466.

HRESI-MS: [M þ Na]^þm/z 466.1952 (calcd 466.1994). Anal.

(C₂₈H₂₉NO₄) C, H, N.

1-(4-Chlorobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl-

3,5-dimethoxybenzoate (12b). 9e (300 mg, 0.96 mmol) and 3,5-

dimethoxybenzoyl chloride (289 mg, 1.44 mmol) were allowed

to react under conditions similar to those used in the prepara-

tion of 1a,b to yield, after purification on a silica gel column

using hexanes-DCM (3:7), 12b (397 mg, 87%) as a yellow oil

that solidified upon standing. ¹H NMR (CDCl₃): δ 1.38 (6H, s),

2.03 (3H, s), 3.87 (6H, s), 4.48 (2H, s), 5.41 (1H, s), 6.19 (1H, d,

J = 9.0 Hz), 6.72-6.77 (2H, m), 6.93 (1H, d, J = 2.4 Hz), 7.29

(4H, m), 7.34 (2H, m). ¹³C NMR (CDCl₃): δ 18.7, 28.5, 47.6,

55.6, 57.2, 106.2, 107.6, 111.9, 116.7, 120.8, 123.8, 127.5, 127.6,

128.7, 130.3, 131.8, 132.3, 137.9, 141.6, 141.6, 160.7, 165.5. ESI-

MS: [MþNa]^þm/z 500. HRESI-MS: [MþH]^þm/z 500.1602 (calcd

500.1599). Anal. (C₂₈H₂₈ClNO₄) C, Cl, H, N.

1-(4-Methylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl-

3,5-dimethoxybenzoate (12c). 9f (300 mg, 1.02 mmol) and 3,5-

dimethoxybenzoyl chloride (307 mg, 1.53 mmol) were allowed

to react under conditions similar to those used in the prepara-

tion of 1a,b to yield, after purification on a silica gel column

using hexanes-DCM (3:7), 12c (393 mg, 84%) as a yellow oil

that solidified upon standing. ¹H NMR (CDCl₃): δ 1.37 (6H, s),

2.00 (3H, s), 2.31 (3H, s), 3.84 (6H, s) 4.47 (2H, s), 5.37 (1H, s),

6.24 (1H, d, J = 9.0 Hz), 6.69-6.73 (2H, m), 6.89 (1H, d, J=2.8

Hz), 7.10 (2H, d, J = 7.5 Hz), 7.22 (2H, d, J=7.5 Hz), 7.31 (2H,

m). ¹³C NMR (CDCl₃): δ 18.9, 21.2, 28.8, 48.1, 55.8, 57.4,

106.4, 107.8, 112.2, 116.6, 121.0, 123.8, 126.3, 127.6, 129.5,

130.5, 132.1, 136.3, 136.5, 141.6, 142.3, 160.9, 165.7. ESI-MS:

[M þ Na]^þm/z 480. HRESI-MS: [M þ Na]^þm/z 480.2152

(calcd 480.2151). Anal. (C₂₉H₃₁NO₄) C, H, N.

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl Octanoate

(13d). 9a (1 g, 3.6 mmol) and octanoyl chloride (878 mg, 5.4

mmol) were allowed to react under conditions similar to those

used in the preparation of 1a,b to yield, after purification on a

silica gel column using hexanes-DCM (85:15), 13d (1.23 g,

84.2%) as a yellow oil. ¹H NMR (DMSO-d₆): δ 0.86 (3H, t, J =

6.9 Hz), 1.26-1.28 (8H, m), 1.33 (6H, s), 1.58 (2H, dd, J₁= J₂=

7.2 Hz), 1.92 (3H, s), 2.47 (2H, t, J=7.2 Hz), 4.49 (2H, s), 5.49

(1H, s), 6.14 (1H, d, J = 8.7 Hz), 6.56 (1H, dd, J₁= 8.7 Hz and

J₂= 2.7 Hz), 6.70 (1H, d, J=2.7 Hz), 7.20-7.31 (5H, m). ¹³

C

NMR (DMSO-d₆): δ 14.4, 18.7, 22.5, 24.9, 28.5, 28.8, 28.9, 31.6,

33.9, 47.8, 57.3, 112.0, 116.8, 121.4, 123.4, 126.5, 126.9, 128.7,

128.9, 131.2, 140.1, 141.3, 141.9, 172.7. ESI-MS: [M þ Na]^þm/z

428. HRESI-MS: [M þ Na]^þm/z 428.2535 (calcd 428.2565).

Anal. (C₂₇H₃₅NO₂) C, H, N.

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ylcyclohex-

anecarboxylate (13e). 9a (2 g, 7.2 mmol) and cyclohexane carbo-

xylic acid chloride (1.57 g, 10.7 mmol) were allowed to react

under conditions similar to those used in the preparation of 1a,b

to yield, after purification on a silica gel column using hexa-

nes-DCM (8:2), 13e (2.5 g, 89.1%) as a yellow oil that solidified

upon standing. ¹H NMR (DMSO-d₆): δ 1.22-1.45 (6H, m), 1.33

(6H, s), 1.54-1.61 (2H, m), 1.67-1.74 (3H, m), 1.92 (3H, s), 4.49

(2H, s), 5.48 (1H, s), 6.14 (1H, d, J = 8.7 Hz), 6.55 (1H, dd, J₁=

8.7 Hz and J₂= 2.7 Hz), 6.69 (1H, d, J = 2.7 Hz), 7.24-7.31

(5H, m). ¹³C NMR (DMSO-d₆): δ 18.7, 22.1, 25.1, 25.8, 29.0,

42.5, 47.8, 57.3, 112.0, 116.8, 121.3, 123.4, 126.5, 126.9, 128.7,

128.9, 131.2, 140.1, 141.4, 141.8, 174.7. ESI-MS: [M þ Na]^þm/z

980 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3

Fotie et al.

412. HRESI-MS: [M þ Na]^þm/z 412.2235 (calcd 412.2252).

Anal. (C₂₆H₃₁NO₂) C, H, N.

(δ 101.4) and C-7 (δ 160.3) were observed but not with C-8 (δ

98.0), proving that C-8 and H-5 are separated by more than

three bonds. H-8 displayed correlations with C-6 (δ 101.4), C-7

(δ160.3), and C-8a (δ 145.9) but not with C-5 (δ 124.6). This

provides evidence that the methoxy group is attached to carbon

C-7. This methoxy group was converted to a 7-hydroxy group

using BBr₃(3 equiv) in DCM (25 mL), with the temperature

being allowed to rise from -78 Cꢀ (1 h) to 25 Cꢀ (18 h)^19,20to

yield 1,2-dihydro-2,2,4-trimethylquinolin-7-ol (276 mg, 45%).

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-7-yl Acetate (17b).

1,2-Dihydro-2,2,4-trimethylquinolin-7-ol (300 mg, 1.6 mmol) was

benzylated at the N1 position with benzyl bromide (410 mg, 2.4

mmol), and the product was further acetylated as described above

to yield 17b (230 mg, 45%) as a yellow oil. ¹H NMR (DMSO-d₆): δ

1.31 (6H, s), 1.95 (3H, s), 2.11 (3H, s), 4.49 (2H, s), 5.40 (1H, s), 5.90

(1H, d, J = 2.7 Hz), 6.26 (1H, dd, J₁= 8.1 Hz and J₂= 2.7 Hz),

6.99 (1H, d, J = 8.1 Hz), 7.15-7.28 (5H, m). ¹³C NMR (DMSO-

d₆): δ 18.8, 21.2, 28.8, 47.5, 57.5, 104.9, 109.0, 120.2, 124.3, 126.4,

126.9, 128.6, 128.8, 129.5, 139.7, 145.5, 151.5, 169.3. ESI-MS: [M þ

H]^þm/z 344. HRESI-MS: [MþH]^þm/z 344.4033 (calcd 344.4026).

Anal. (C₂₁H₂₃NO₂) C, H, N.

1,2-Dihydro-2,2-dimethylquinolin-6-ol (19). A mixture of p-

phenetidine (4.0 g, 29.2 mmol), 3-chloro-3-methyl-1-butyne (3.6

g, 35.1 mmol), CuCl (287 mg, 2.9 mmol), and Cu (184 mg, 2.9

mmol) in Et₃N (75 mL) was allowed to stir at room temperature,

and the reaction was monitored by TLC until completion (4-

5 h).¹⁸The reaction mixture was dissolved in CH₂Cl₂and

washed twice with 200 mL of a saturated solution of NH₄Cl.

The solvent was then evaporated and the residue purified on a

silica gel column using hexanes-EtOAc (95:5) to yield 4-ethoxy-

N-(2-methylbut-3-yn-2-yl)benzenamine (18) (3.26 g, 55%) as a

yellow oil. 18 (3.26 g, 16.0 mmol) and CuCl (159 mg, 1.6 mmol)

in toluene was heated to reflux overnight.^18,21,22The solvent was

then evaporated and the residue purified on a silica gel column

using hexanes-ethyl acetate (9:1) to yield 6-ethoxy-1,2-dihydro-

2,2-dimethylquinoline (2.10 g, 65%) that was subsequently

heated to reflux in HBr for 16 h to yield 1,2-dihydro-2,2-

dimethylquinolin-6-ol (19) (1.6 g, 88.4%), mp 155-157 ꢀC. ¹H

NMR (DMSO-d₆): δ 1.17 (6H, s), 5.15 (1H, brs, NH), 5.43 (1H,

d, J = 9.6 Hz), 6.11 (1H, d, J = 9.6 Hz), 6.29 (1H, d, J = 9.3 Hz),

6.31-6.38 (2H, m), 8.30 (1H, brs, OH). ¹³C NMR (DMSO-d₆): δ

30.5, 51.7, 113.2, 113.7, 115.7, 120.6, 123.9, 132.4, 137.3, 148.4.

ESI-MS: [M þ Na]^þm/z 198.

1-Acetyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl Acetate (15a).

Compound 4 (500 mg, 2.64 mmol), excess acetyl chloride (5 equiv),

and NaH (3 equiv) in THF were heated to reflux overnight under

nitrogen. The solvent was then evaporated and the residue parti-

tioned between water and DCM, and purified on a silica gel column

using hexanes-DCM (1:1) to yield 14a (560 mg, 77.6%) as a yellow

oil. ¹H NMR (DMSO-d₆): δ 1.44 (6H, s), 1.96 (3H, s), 2.08 (3H, s),

2.26 (3H, s), 5.66 (1H, s), 6.93 (1H, d, J = 8.6 Hz), 6.95-6.96 (2H,

m). ¹³C NMR (DMSO-d₆): δ 17.8, 21.3, 26.1, 26.7, 57.8, 116.7,

120.7, 124.4, 127.2, 129.7, 134.4, 136.9, 147.5, 169.7, 171.9. ESI-MS:

[M þ Na]^þm/z 296. HRESI-MS: [M þ Na]^þm/z 296.1258 (calcd

296.1263). Anal. (C₁₆H₁₉NO₃) C, H, N.

1,2-Dihydro-1,2,2,4-tetramethylquinolin-6-yl Acetate (15b).

1,2-Dihydro-2,2,4-trimethylquinolin-6-yl acetate (14) was ob-

tained by acetylation of 4. ¹H NMR (DMSO-d₆): δ 1.19 (6H, s),

1.84 (3H, s), 2.18 (3H, s), 5.32 (1H, s), 5.84 (1H, brs, NH), 6.40

(1H, d, J = 7.8 Hz), 6.61-6.64 (2H, m). Compound 14 (562 mg,

2.4 mmol), CH₃I (756 μL, 12.1 mmol), and Et₃N (507 μL, 3.6

mmol) in toluene (25 mL) were heated to reflux overnight. The

solvent was then evaporated and the residue was purified on a

silica gel column using hexanes-ethyl acetate (9:1) to yield

1,2-dihydro-1,2,2,4-tetramethylquinolin-6-yl acetate (15b) (495

mg, 84%) as a yellow oil. ¹H NMR (CDCl₃): δ 1.32 (6H, s), 1.97

(3H, s), 2.29 (3H, s), 2.80 (3H, s), 5.35 (1H, s), 6.48 (1H, d, J =

8.7 Hz), 6.79 (1H, d, J = 2.7 Hz), 6.84 (1H, dd, J₁= 8.7 Hz and

J₂= 2.7 Hz). ¹³C NMR (CDCl₃): δ 18.5, 21.1, 27.1, 30.8, 56.3,

110.8, 116.3, 120.9, 124.0, 127.6, 131.1, 141.4, 143.1, 170.2. ESI-

MS: [M þ H]^þm/z 268. HRESI-MS: [M þ H]^þm/z 268.1302

(calcd 268.1313). Anal. (C₁₅H₁₉NO₂) C, H, N.

1,2-Dihydro-2,2,4-trimethylquinoline (16a). This compound

was prepared by heating a mixture of aniline (1 g, 10.7 mmol)

and an excess of acetone (5 mL) in toluene (25 mL) to reflux in

the presence of iodine (272 mg, 1.07 mmol) for 24 h.^14-16The

solvent was then evaporated and the residue purified on a silica

gel column using hexanes-DCM (9:1) to yield 1,2-dihydro-

2,2,4-trimethylquinoline (16a) (538 mg, 29%). ¹H NMR

(CDCl₃): δ 1.41 (6H, s), 2.19 (3H, s), 3.74 (1H, brs, NH), 5.46

(1H, s), 6.57 (1H, d, J=7.8 Hz), 6.81 (1H, dd, J₁=J₂= 7.8 Hz),

7.15 (1H, dd, J₁=J₂=7.8 Hz), 7.23 (1H, d, J=7.8 Hz). ¹³C NMR

(CDCl₃): δ 18.7, 31.2, 51.9, 113.1, 117.2, 121.6, 123.8, 128.5,

128.6, 143.5.

1-Benzyl-1,2-dihydro-2,2,4-trimethylquinoline (17a). 1,2-Di-

hydro-2,2,4-trimethylquinoline (16a) (300 mg, 1.7 mmol) was

benzylated at the N1 position as described above with benzyl

bromide (444 mg, 2.6 mmol) to yield, after purification on a

silica gel column using hexanes-DCM (7:3), 17a (409 mg,

91.3%) as a yellow oil that solidified upon standing. ¹H NMR

(CDCl₃): δ 1.42 (6H, s), 2.09 (3H, s), 4.56 (2H, s), 5.38 (1H, s),

6.30 (1H, d, J = 8.4 Hz), 6.64-6.66 (1H, m), 6.93-6.96 (1H, m),

7.13 (1H, d, J = 7.5 Hz), 7.25-7.41 (5H, m). ¹³C NMR (CDCl₃):

δ 18.8, 28.7, 48.0, 57.1, 111.8, 116.0, 122.7, 123.5, 126.2, 126.5,

127.9, 128.6, 129.3, 139.7, 144.1. ESI-MS: [M þ Na]^þm/z 286.

HRESI-MS: [M þ Na]^þm/z 286.1583 (calcd 286.1572). Anal.

(C₁₉H₂₁N) C, H, N.

1-Benzyl-1,2-dihydro-2,2-dimethylquinolin-6-ol (20a). 19 (300

mg, 1.7 mmol) was benzylated at the N1 position with benzyl

bromide (445 mg, 2.6 mmol) as described above to yield, after

purification on a silica gel column using hexanes-ethyl acetate (9:1),

20a (341 mg, 76%) as purple crystals, mp 162-165 ꢀC. ¹H NMR

(DMSO-d₆): δ 1.30 (6H, s), 4.38 (2H, s), 5.56 (1H, d, J =9.6 Hz),

6.01 (1H, d, J = 8.7 Hz), 6.22 (1H, d, J = 9.6 Hz), 6.28 (1H, dd, J₁

= 8.7 Hz and J₂= 2.4 Hz), 6.37 (1H, d, J₂= 2.4 Hz), 7.17-7.31

(5H, m), 8.44 (1H, brs, OH). ¹³C NMR (DMSO-d₆): δ 27.7, 47.8,

57.1, 112.9, 114.0, 115.2, 122.8, 123.7, 126.6, 126.8, 128.8, 133.5,

137.1, 141.0, 148.7. ESI-MS: [M þ Na]^þm/z 288. HRESI-MS: [M

þ H]^þm/z 288.1354 (calcd 288.1364). Anal. (C₁₈H₁₉NO) C, H, N.

1-(3,5-Dimethoxybenzyl)-1,2-dihydro-2,2-dimethylquinolin-6-

yl Acetate (20b). 19 (300 mg, 1.7 mmol) was benzylated at the N1

position with 3,5-dimethoxybenzyl bromide (601 mg, 2.6 mmol)

as described above to yield a brown oil (446 mg, 81%) that was

further acetylated to yield, after purification on a silica gel

column using hexanes-ethyl acetate (9:1), 20b (453 mg, 90%)

as a yellow oil that solidified upon standing. ¹H NMR (DMSO-

d₆): δ 1.33 (6H, s), 2.17 (3H, s), 3.69 (6H, s), 4.41 (2H, s), 5.60

(1H, d, J = 9.6 Hz), 6.13 (1H, d, J = 8.4 Hz), 6.28 (1H, d, J =

9.6 Hz), 6.30-6.34 (1H, m), 6.45-6.47 (2H, m), 6.57 (1H, d, J =

8.4 Hz), 6.43-6.45 (1H, m). ¹³C NMR (DMSO-d₆): δ 21.2, 28.5,

47.9, 55.5, 57.6, 98.2, 104.6, 112.0, 119.8, 121.5, 121.9, 122.9,

133.4, 141.4, 141.8, 142.8, 161.1, 170.0. ESI-MS: [M þ Na]^þm/z

390. HRESI-MS: [M þ Na]^þm/z 390.1665 (calcd 390.1681).

Anal. (C₂₂H₂₅NO₄) C, H, N.

1,2-Dihydro-7-methoxy-2,2,4-trimethylquinoline (16b). This

compound was prepared by heating to reflux a mixture of m-

anisidine (1 g, 8.1 mmol) and an excess of acetone (5 mL) in

toluene (25 mL) in the presence of iodine (206 mg, 0.81 mmol)

1

for 24 h to yield 16b (660 mg, 40%) as a yellow oil. H NMR

(DMSO-d₆): δ 1.18 (6H, s), 1.85 (3H, s), 3.65 (3H, s), 5.11 (1H, s),

5.79 (1H, brs, NH), 6.01-6.05 (2H, m), 6.83 (1H, d, J = 7.8 Hz).

¹³C NMR (DMSO-d₆): δ 18.7, 31.4, 51.6, 55.0, 98.0, 101.4,

114.5, 124.6, 126.2, 127.7, 145.9, 160.3. Molecular formula:

C₁₃H₁₇NO. The chemical shifts of protons and carbons of this

compound were attributed using a HSQC spectrum, and the

structure of the compound was further confirmed by key

correlations observed in the HMBC spectrum. Correlations

between H-5 (δ 6.83) and C-4 (127.7) and between C-6

Article

Quantum Chemical Calculations. Quantum chemical optimi-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 981

Solomon Nwaka of WHO/TDR, Reto Brun of STI, and

Xiaohua Zhu of The Ohio State University for their contribu-

tions to the project and Don Patrick (University of North

Carolina) for a critical reading of the manuscript.

zation calculations were performed using Jaguar, version 7.6,

installed on an AMD Opteron 64 bit dual core workstation with

4GB RAM running the openSuse 10.1 operating system. Den-

sity functional theory with B3LYP hybrid functionals was used

to perform the calculation with a 6-31þG** basis set. Jaguar’s

standard Poisson-Boltzmann continuum solvation model

(PBF) was implemented in the calculation with water as solvent.

Initial compound coordinates were built on Maestro, version 9.0

(Schrodinger Suite 2009), modeling suite environment. Mole-

cular mechanics conjugate gradient method was applied to

minimize the geometries utilizing OPLS2005 force field

(maximum iteration 500, gradient 0.05). Jaguar calculations

were performed on conjugated and open shell models of each

compound.

Supporting Information Available: Scatter plot of IC₅₀values

vs ClogP for monomeric dihydroquinolines containing a 6-

hydroxyl group or a 6-O-acylated function (Figure S1) and

quantum chemical calculations for 4, 6a, 9a, and the dihydro-

quinolin-6-ols corresponding to 15a and 15b (Table S1). This

material is available free of charge via the Internet at http://

pubs.acs.org.

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In Vitro Susceptibility Testing of Trypanosomes and L6 Myo-

blasts. The in vitro antitrypanosomal activity and cytotoxicity of

compounds of interest were determined following established

protocols.^23,24,57

In Vivo Assay. Compounds were evaluated in mice infected

with Trypanosoma b. brucei (STIB 795) following an acute in

vivo model developed at STI. In brief, the tested compounds

were first dissolved in 100% DMSO followed by dilution in

water to a final concentration of 10% DMSO. Female NMRI

mice (20-25 g) were kept in standard Macrolon cages at a

constant temperature of 21 ꢀC and 60-70% relative humidity

and were provided commercial pellets and water ad libitum. On

day 0 for each group (control as well as experimental), four mice

were infected intraperitoneally with 1 ꢀ 10⁴bloodstream form

T. b. brucei from a stock of cryopreserved stabilates containing

10% glycerol. The stabilate was suspended in phosphate/saline/

glucose buffer (9.0 g/L Na₂HPO₄2H₂O, 0.47 g/L NaH_2-

3

PO₄2H₂O, 2.55 g/L NaCl, 1% w/v glucose, pH 8.0) to obtain

3

a trypanosome cell density of 4 ꢀ 10⁴/mL. Each mouse was

injected with 0.25 mL. On days 3-6, animals were treated on

4 consecutive days with the compound of interest by the

intraperitoneal route, with a tolerated dose that was determined

earlier. The concentration of test compound is such that 0.1 mL/

10 g can be injected. On day 7, parasitemia of all mice is checked

by tail blood examination and recorded as well as on day 10.

After day 10, parasitemia is monitored twice per week until day

30. For dead mice, the day of death is recorded. On day 30,

surviving and aparasitaemic mice are considered cured, and they

are later euthanized.

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TDR through Grant A50866. We thank Alan Hudson and

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Article Doi

Antitrypanosomal activity of 1,2-dihydroquinolin-6-ols and their ester derivatives

DOI: 10.1021/jm900723w

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